Compositions and methods for regulating an immune response in a subject

ABSTRACT

The present invention relates to compositions and methods for regulating an immune response in a subject, particularly to treat a subject with a tumor, notably a solid tumor, or an infectious disease. Disclosed are methods of regulating the innate immunity in a subject, such as by regulating the activity of γδ T cells in a subject. Disclosed are combinations of particular agents, such as a cytokine and a γδ T cell activator, particular regimens and dosages can produce a remarkable expansion of γδ T cells in vivo and a remarkable increase in a subject&#39;s immune defense. The invention can be used for therapeutic purposes, to produce, regulate or facilitate an immune response in a subject.

FIELD OF THE INVENTION

The present invention relates to compositions an methods for regulatingan immune response in a subject, particularly a T cell response in asubject. The present invention more specifically discloses efficientmethods of regulating the innate immunity in a subject, such as byregulating the activity of γδ T cells in a subject. The inventionfurther provides that said methods and compounds may be used in thetreatment of solid tumors and particularly tumors involving metastases.

BACKGROUND

Most human peripheral blood y T cells express a γδTCR heterodimerencoded by Vγ9/Vδ2 genes, some NK-lineage receptors for MHC class I andalmost no CD4 nor CD8. These cells have been shown to exhibit strong,non MHC-restricted, cytolytic activity against virus-infected cells(Poccia et al (1999), parasite-infected cells (Constant et al (1995)),or tumor cells (Fournie et Bonneville (1996)). These cells are alsophysiologically amplified in the context of several unrelated infectiousdiseases such as tuberculosis, malaria, tularemia, colibacillosis andalso by B-cell tumors (for review see Hayday, 2000).

Beside their anti-infectious activity, it was shown in short termcytotoxicity assays that Vγ9/Vδ2 T cells are able to lyse a wide varietyof tumor cell lines from very diverse origins: lymphoma and leukemiafrom B-cell, T-cell or myeloid lineages (Fisch et al., 1997; Selin etal., 1992; Sicard et al., 2001; Sturm et al., 1990; Zheng et al.,2001a), breast carcinoma (Bank et al., 1993), glioblastoma (Fujimiya etal., 1997; Yamaguchi et al., 1997), renal cell carcinoma (Choudhary etal., 1995; Kobayashi et al., 2001; Mitropoulos et al., 1994),nasopharyngeal carcinoma (Zheng et al., 2001b), lung adenocarcinoma(Ferrarini et al., 1996).

In microbes, Vγ9/Vδ2⁺ lymphocytes spontaneously recognize a structurallyrelated set of nonpeptide antigens, referred to as naturalphosphoantigens and alkylamines. In B cell tumors, the nature ofantigens for the γδ T cells remains unidentified. Vγ9/Vδ2⁺ lymphocytesare also responsive to a variety of virally infected-, activated- ortumoral cell types without prior exposure. Again, in these situations,the responsible antigens remain unknown (for review see Fisch, 2000). Ithas been shown that, in vitro, Vγ9/Vδ2⁺ lymphocytes respond to syntheticdrugs such as therapeutic aminobisphosphonates (reviewed in Espinosa,2001), leading to their in vitro activation. Recognition of naturalnon-peptide antigens is mediated by the γδ TCR, through amino acidresidues located on both Vγ9- and Vδ2-CDR3 regions. Although neitherprocessing nor presentation by CD1 or MHC molecules is involved,Vγ9/Vδ2⁺ lymphocyte activation by non-peptide antigens appears torequire cell-to-cell contact (Lang, 1995 ; Morita, 1995 ; Miyagawa, 2001, Rojas, 2002).

The stimulating bacterial antigens have been shown to be small nonpeptidic compounds classically referred to as phosphoantigens (Bohr etal., 1996; Belmant et al., 2000; Constant et al., 1994; Poquet et al.,1998; Tanaka et al., 1995), owing to the presence of phosphate groups inmost instances.

Endogenous Metabolites of the Mevalonate Pathway: IPP

Vγ9/Vδ2 T cells can also be activated through endogenous metabolites(acting in the micromolar range) such as isopentenyl pyrophosphate orIPP (Espinosa et al., 2001b; Tanaka et al., 1995), which is producedthrough the conventional mevalonate pathway shared by bothmicroorganisms and mammalian cells. Production of IPP in the lattercells can be up-regulated in situations of cell stress andtransformation. In particular a recent study has reported a correlationbetween the endogenous production levels of IPP in tumor cells and theirsusceptibility to Vγ9/Vδ2 T cell-mediated lysis (Gober et al., 2003).

Compounds Regulating Endogenous Metabolites: Statins andAminobisphosphonates

Also consistent with a direct contribution of endogenous metabolites ofthe mevalonate pathway to Vγ9/Vδ2 T cell recognition, cell treatmentwith pharmacological agents preventing IPP biosynthesis (such asstatins) or leading to IPP accumulation (such as aminobisphosphonates,see below) lead respectively to decreased or enhanced Vγ9/Vδ2 T cellstimulating properties of the treated cells (Gober et al., 2003; Kato etal., 2001).

Aminobisphosphonates are thought to inhibit FPP synthase, an enzyme inthe mevalonate pathway, the inhibition of which causes the accumulationand release of upstream isoprenoid lipids such as IPP.Aminobisphosphonate compounds had been used in human therapy for thetreatment of bone metastases in cancer patients, and provided a firstset of evidence for in vivo expansion of human Vγ9/Vδ2⁺ lymphocytesinduced by phosphoantigen agonists, reporting increases of circulatingγδ T cells within one to three weeks in human adults with multiplemyeloma after therapeutic intravenous injection of 60-90 mg ofpamidronate (Kunzmann et al, 1999). However, such compounds requirepresentation by antigen presenting cells and cannot produce substantialstimulation of Vγ9/Vδ2 T cell activity as assessed by cytokine secretionin a pure Vγ9//Vδ2 T cell culture. Moreover, pamidronate shows very lowpotency of activation of γδ T cells, reported to achieve at best only2-fold increase in γδ T cell count (Wilhelm et al., 2003).

High Specific Activity Phosphoantigens

Recently, several highly potent γδ T cell activatingpyrophosphate-containing compounds have been described which directlyactivate γδ T cells. In particular, phosphalyhydrin and phosphoepoxydecompounds were described by the group of J. J. Fournie. (R,S)-3-(bromomethyl)-3-butanol-1-yl-diphosphate, also referred to as BrHPP(BromoHydin PyroPhosphate) is currently used in ongoing human clinicalstudies to stimulate the proliferation of γδ T cells ex vivo. Otherpyrophosphate containing compounds with high specific activity (EC50 inthe nanomolar or better range) are produced through an isoprenoidbiosynthetic pathway called the “Rohmer” or “non-mevalonate” pathway,which is specific to pro- and eukaryotic microorganisms (Feurle et al.,2002; Jomaa et al (2003); Jomaa et al., 1999a; Jomaa et al., 1999b;Rohmer et al., 1993).

In contrast to aminobisphosphonates and statins discussed above, highspecific activity phosphoantigen compounds such as the compounds offormula I to formula XVII are capable of regulating Vγ9/Vδ2 T cellactivity in a population of Vγ9/Vδ2 T cell clones in culture atmillimolar concentrations, where regulation is assessed by monitoringcytokine secretion. While the precise mode of recognition ofphosphoantigens remains unclear, a direct contribution of the Vγ9/Vδ2TCR to phosphoantigen-mediated activation has-been demonstrated by genetransfer experiments (Bukowski et al., 1995). Accordingly recentstructural data drawn from crystallographic analysis of Vγ9/Vδ2 TCR arecompatible with cognate interactions between phosphoantigens and γδ TCR,through electrostatic interactions between the negatively chargedphosphate residues on the antigen side with several positively chargedamino-acids on the TCR side (Allison et al., 2001).

Methods of Treatment Involving Administration of γδ T Cell ActivatingCompounds

Despite the foregoing, studies of phosphoantigens including thesynthesis and in vitro testing of analogs from a variety of groups ofcompounds indicate structures providing high γδ T cell activation, inparticular compounds according to formula I described herein. However,no methods or treatment regimens have been proposed for the use ofphosphoantigens with high specific activity in vivo. Accordingly, nomethods or treatment regimens have been proposed for strategiesinvolving an in vivo stimulation sufficient to generate a large increasein γδ T cell activity.

In one aspect, research into treatment regimens based on γδ T cellactivating compounds has been hampered by the lack of suitable in vivomodels. Evidence for a general immune surveillance function of theinnate immune system has been provided by various in vivo models: micedeficient in innate effector cells such as NK cells, NKT cells or γδ Tcells show a significantly increased incidence of tumors (Girardi etal., 2001; Kim et al., 2000; Smyth et al., 2000). However, such resultscan only be transposed to the human situation with caution, as thesecell populations are somewhat different in humans as compared to mice.In particular, the human Vγ9V/δ2 cell population for example does nothave a formal equivalent in rodents.

In view of the foregoing, although several compounds have been shown tohave in vitro activity, their in vivo activity and more generally the invivo kinetics of γδ T T cells in response to stimulation had not beenexplored. Accordingly, efficient methods are needed to selectivelyactivate γδ T cells in vivo, in a subject, under conditions suitable fortherapy.

Furthermore, no therapeutic strategy involving stimulating a manifoldincrease of circulating γδ T cells in vivo had been developed for thetreatment of tumors, and in particular for solid tumors, especiallythose with metastases. The safety and efficacy of treatments for tumorscan be altered by a variety of factors, and treatments can be affectedby tumor growth kinetics, drug resistance of tumor cells, total-bodytumor cell burden, toxic effects of therapy on cells and tissues otherthan the tumor, and distribution of therapeutic agents within thetissues of the patient. The greater the size of the primary tumor, thegreater the probability that a large number of cells (drug resistant anddrug sensitive) have metastasized before diagnosis and that the patientwill relapse. Solid tumors and carcinomas account for more than 90% ofall cancers in man, and although the use of monoclonal antibodies andimmunotoxins has been investigated in the therapy of lymphomas andleukemias, many such agents have been disappointingly ineffective inclinical trials against carcinomas and other solid tumors. One possiblereason for the ineffectiveness of effector-cell-based treatments is thatcells are not readily transported into solid tumors. Alternatively, evenonce within a tumor mass, these cells may fail to distribute evenly dueto the presence of tight junctions between tumor cells, fibrous stroma,interstitial pressure gradients and binding site barriers.

SUMMARY OF THE INVENTION

The present invention now discloses particular compositions and methodsthat can be used to efficiently regulate the activity of γδ T cells,particularly the activation and proliferation of γδ T cells, in vivo ina subject. These compositions and methods are particularly suited forimmuno-therapy in a subject, particularly in a subject having a tumorand more particularly a subject having a solid tumor. Nevertheless, theinvention may also be useful for therapy of a subject suffering fromother diseases, particularly an infectious disease.

The compositions and methods provided herein by the inventors are basedon a series of results. In one aspect, a therapeutic strategy usingautologous γδ T cells activated ex-vivo by a high specific activitypyrophosphate compound shows indications of anti-tumor activity in humanpatients in an ongoing clinical study using ex-vivo stimulated γδ Tcells for the treatment of metastatic renal cell carcinoma. In anotheraspect, the compositions and methods according to the invention arebased on a series of findings resulting from the first known experimentsin animals involving regulating the activity of γδ T cells, includingboth in vivo increase of the biological activity of γδ T cells as wellas manifold expansion of the γδ T cell population. Furthermore, in anovel Nod-Scid murine model adapted for the assessment of γδ T cellactivation and γδ T cell mediated anti-tumor activity, it has been foundthat high potency γδ T cell activating compounds administered to theanimal can regulate y8 T cell activity in vivo, that γδ T cells caninfiltrate solid tumors, and moreover that such treatment is effectivein decreasing the mass of solid tumors, and more particularly metastatictumors. The anti-tumoral effect of γδ T cell activator-stimulate γδ Tcells was also observed toward fresh cells in culture obtained fromhuman patients having metastatic solid tumors, but was not observedtowards non-tumoral cells from the same patients. Based on suchdiscoveries, the inventors have devised therapies for solid tumors usingcompounds capable of regulating the activity of γδ T cells.

In further experiments, in vivo kinetics of high specific activity γδcell activators was determined. This provided methods for administeringand using such compounds for the treatment of a wide range ofapplications for which modulating of the immune response is desired,including for the treatment or prevention of infection, autoimmunedisorders, tumors. More specifically, the elucidation of in vivo γδ Tcell kinetics resulted in the following findings, among others:

-   -   (a) that the activity of γδ T cells may be regulated repeatedly,        including activating cells as demonstrated by cytokine release        and expansion of the cell population, using a γδ cell activator,        depending on the administration regimen, and that certain        intervals of drug administration provide for optimal        re-stimulation of γδ T cell activity;    -   (b) that addition of a cytokine in an administration regimen,        particularly II-2, and more particularly certain doses of IL-2        provide improved in vivo expansion of γδ cells    -   (c) methods for translating in vitro activity to in vivo dosage        regimens for γδ T cell activator capable of increasing γδ cell        activity;    -   (d) specific dosage and administration regimens allowing the        increase of γδ cell activity using γδ T cell activators.

In one aspect, the invention discloses a method for treating a tumor,said method comprising the step of administering, in at least onetreatment, a therapeutically effective amount of a γδ T cell activator,together with a pharmaceutically acceptable carrier, to a warm-bloodedanimal in need of such treatment. In a particularly preferred aspect,provided is a method for treating a solid tumor, said method comprisingthe step of administering, in at least one treatment, a therapeuticallyeffective amount of a γδ T cell activator, together with apharmaceutically acceptable carrier, to a warm-blooded animal in need ofsuch treatment. Also provided is a method for treating a solid tumor,said method comprising the step of administering, in at least onetreatment, a therapeutically effective amount of a γδ T cell activator,together with a pharmaceutically acceptable carrier, to a warm-bloodedanimal having a solid tumor with metastases in need of such treatment.Said methods of treating a tumor can be carried out in any suitablefashion. Preferably said methods comprise the step of contacting a γδ Tcell in a warm-blooded animal having a solid tumor, with atherapeutically effective amount of a γδ T cell activator, or optionallysaid methods comprise the step of providing in the bloodstream of awarm-blooded animal having a solid tumor a therapeutically effectiveamount of a γδ T cell activator. In a particular embodiment, said tumoris a solid tumor with metastases. Alternatively, said tumor is ahaematological tumor, preferably a lymphoma. Optionally, said tumor is ametastatic tumor. Preferably, said tumor is selected from the groupconsisting of lung, colorectal, prostate, breast or epidermoid head orneck tumors. In a preferred aspect of the invention, said tumor is arenal cancer, preferably a metastatic renal cancer. Alternatively, saidtumor is selected from the group consisting of a melanoma, ovariancancer, pancreas cancer, neuroblastoma, head or neck cancer, bladdercancer, renal cancer, brain cancer and gastric cancer. Preferably, saidmethod comprises at least two treatments. Preferably, said γδ T cellactivator is administered with an interval of about two weeks to abouteight weeks between treatments, more preferably with an interval ofabout three to about four weeks between treatments. Optionally, at leastthree, four or six treatments are administered to said animal.Preferably, the biological activity of γδ T cells are increased in saidwarm-blooded animal. Preferably, the number of circulating γδ T cellsare increased in said warm-blooded animal. In a particular embodiment,the amount of said γδ cell activator is sufficient to expand the γδ cellpopulation in a subject to reach at least 30%, 40%, 50% or 60%, orbetween 30-90% of total circulating lymphocytes. In an other particularembodiment, the amount of said γδ T cell activator is sufficient toinduce an at least 10-fold increase in the γδ T cell population in asubject. Preferably, said γδ cell population is assessed between day 4and day 8 following administration of said γδ T cell activator, morepreferably at day 5, 6 or 7 following administration of said Uγδ T cellactivator. Preferably, said γδ T cell population is assessed by flowcytometry. Preferably, said γδ T cells are Vγ9/Vδ2 T cells. Optionally,said γδ T cell activator is administered by intravenous infusion,preferably said infusion takes place during about 5 to about 120 min,more preferably during about 5 to about 30 min. In further preferredaspects, the methods may comprise further administering a cytokine,preferably IL2.

In another aspect, disclosed is a method treating a tumor disease in awarm-blooded animal, said method comprising administering, in more thanone treatment, a therapeutically effective amount of a γδ cellactivator, together with a pharmaceutically acceptable carrier, to awarm-blooded animal in need of such treatment, wherein the γδ T cellactivator is administered in more than one treatment with an interval ofabout two weeks to about eight weeks between treatments. Also disclosedis a method for stimulating a γδ T cell in a warm-blooded animal, saidmethod comprising: (a) contacting a γδ T cell in a warm-blooded animalwith a therapeutically effective amount of a γδ T cell activator; and(b) repeating step (a) at least once within about two weeks to abouteight weeks after said contacting in step (a). Also disclosed is amethod for stimulating a γδ T cell in a warm-blooded animal, said methodcomprising: (a) providing in the bloodstream of a warm-blooded animal atherapeutically effective amount of a γδ T cell activator; and (b)repeating step (a) at least once within about two weeks to about eightweeks after said contacting in step (a). Optionally, step (b) is saidmethods may comprise repeating step (a) at least twice, at least threetimes, at least four times or at least six times.

The inventors also disclose a method for treating a solid tumor, saidmethod comprising the step of contacting a γδ T cell in a warm-bloodedanimal having a solid tumor with a γδ T cell activator in an amountsufficient to expand the γδ T cell population in a subject to reach atleast 30%, 40%, 50% or 60%, or between 30-90%, of total circulatinglymphocytes. Also provided is method for treating a solid tumor, saidmethod comprising the step of providing in the bloodstream of awarm-blooded animal having a solid tumor a γδ cell activator in anamount sufficient to expand the γδ T cell population in a subject toreach at least 30%, 40%, 50% or 60%, or between 30-90%, of totalcirculating lymphocytes. Preferably the γδ T cell population is assessedbetween day 4 and day 8, most preferably at about day 5, day 6 or day 7,following administration of the a γδ T cell activator.

One aspect of the invention is to increase the γδ T cell population. Theinvention encompasses a method for treating a solid tumor, said methodcomprising the step of contacting a γδ T cell in a warm-blooded animalhaving a solid tumor with a γδ T cell activator in an amount sufficientto induce an at least 10-fold increase in the γδ T cell population in asubject. Also encompassed is a method for treating a solid tumor, saidmethod comprising the step of providing in the bloodstream of awarm-blooded animal having a solid tumor a γδ T cell activator in anamount sufficient to induce an at least 10-fold increase in the γδ Tcell population in a subject compared to the level prior to treatment.Preferably the γδ T cell population is assessed between day 4 and day 8,most preferably at about day 5, day 6 or day 7, following administrationof the a γδ T cell activator. Preferably the γδ T cell population isassessed by flow cytometry.

Also encompassed by the invention is the use of an γδ T activator forthe manufacture of a pharmaceutical preparation for the treatment of atumor, comprising admixing said γδT activator with a pharmaceuticallyacceptable carrier, said pharmaceutical preparation being administeringto said subject. Preferably, said tumor is a solid tumor. In aparticular embodiment, said tumor is a solid tumor with metastases.Alternatively, said tumor is a haematological tumor, preferably alymphoma. Optionally, said tumor is a metastatic tumor. Preferably, saidtumor is selected from the group consisting of lung, colorectal,prostate, breast or epidermoid head or neck tumors. In a preferredaspect of the invention, said tumor is a renal cancer, preferably ametastatic renal cancer. Alternatively, said tumor is selected from thegroup consisting of a melanoma, ovarian cancer, pancreas cancer,neuroblastoma, head or neck cancer, bladder cancer, renal cancer, braincancer and gastric cancer. Preferably, said pharmaceutical preparationis administered at least twice, more preferably with an interval ofabout two weeks to about eight weeks between treatments, still morepreferably with an interval of about three to about four weeks betweentreatments. Optionally, said pharmaceutical preparation is administeredis administered at least three, four or six times. Preferably, saidpharmaceutical preparation increases the biological activity of γδ Tcells in said subject. Preferably, said pharmaceutical preparationincreases the number of circulating γδ T cells in said subject. In aparticular embodiment, the amount of said γδ T cell activator issufficient to expand the γδ T cell population in a subject to reachbetween 30-90% of total circulating lymphocytes. In an other particularembodiment, the amount of said γδ T cell activator is sufficient toinduce an at least 10-fold increase in the γδ T cell population in asubject. Preferably, said γδ T cell population is assessed between day 4and day 8 following administration of said γδ T cell activator, morepreferably at day 7 following administration of said γδ T cellactivator. Preferably, said γδ T cell population is assessed by flowcytometry. Preferably, said γδ T cells are Vγ9/Vδ2 T cells. Optionally,said pharmaceutical preparation is administered by intravenous infusion,preferably said infusion takes place during about 5 to about 120 min,more preferably during about 5 to about 30 min. In further preferredaspects, the methods may comprise further administering a cytokine,preferably IL-2.

In one aspect of the methods of the invention, a γδ T cell activator isadministered in the absence of administration of a cytokine. In otheraspects the methods if the invention comprise the use of a γδ T cellactivator and an interleukin-2 polypeptide, for the manufacture of apharmaceutical composition for regulating the activity of γδ T cells ina mammalian subject, the γδ T cell activator and interleukin-2polypeptide being administered separately to the subject.

In preferred aspects of the any of the methods described herein, atleast two, three, four or six treatments are administered to saidanimal. In preferred aspects of any of the methods described herein, theγδ T cell activator is administered in more than one treatment with aninterval of about two to about eight weeks between treatments, or yetmore preferably about three to about four weeks between treatments.

In any of the method described herein, the warm-blooded animal in thepresent methods may be a rodent or a non-human primate. In preferredaspects, the warm blooded animal in the present methods is a human.

In preferred aspects of any of the aspects of the present invention, themethods result in an increase in the biological activity of γδ T cellssaid warm-blooded animal or said subject, or an increase in the numberof circulating γδ T cells in said warm-blooded animal or said subject.Preferably, the γδ T cells referred to in the methods of the inventionare Vγ9/Vδ2 T cells.

As described in the examples, the methods of the invention may be usedfor the treatment of a solid tumor as well as a solid tumor withmetastases. In other aspects, the methods of the invention may be usedin the treatment of a haematological tumor. Preferably the γδ Tactivator is administered to a human in need of such treatment in a dosethat is appropriate for the treatment of said disease. However,preferred and particularly effective doses are further provided herein.Optionally, in any of the methods described herein, a γδ T activator isadministered in a dose that is greater than the EC50 human dose; and oneor more further doses each greater than the EC50 human dose areadministered in at least one additional treatment after an intervalbetween the treatments of two to eight weeks.

In any of the methods provided herein, the invention provides furtheradministering a cytokine. In preferred aspects of any of the methodsdescribed herein, the cytokine is IL-2. Preferably, the interleukin-2polypeptide is administered at low doses. In further aspects of any ofthe methods of the invention, the cytokine, and most preferably aninterleukin-2 polypeptide, is administered over a period of timecomprised between 1 and 10 days. Preferably, the interleukin-2polypeptide is administered at a daily dose comprised between 0.2 and 2MU per day, even more preferably between 0.2 and 1.5 MU, furtherpreferably between 0.2 and 1 MU. The daily dose of cytokine, preferablyan interleukin-2 polypeptide, is administered as a single injection orin two injections. Preferably the γδ T cell activator is administered asa single dose at the beginning of the treatment.

In preferred aspects, provided is a method for stimulating a γδ T cellin a subject, or a method of treating a cancer, an infectious disease,an autoimmune disease or an allergic disease in a subject, comprising:separately administering to a subject in need thereof an effectiveamount of a γδ T activator and a cytokine, preferably an interleukin-2polypeptide, over a period of time comprised between 1 and 10 days.Preferably the γδ T cell activator is administered as a single dose atthe beginning of the treatment. Preferably the γδ T cell activator isadministered as a single dose at the beginning of the treatment and thecytokine, preferably IL-2, is administered on at least two, three, fouror five days within the ten day period following administration of theγδ T cell activator. The invention also encompasses a product comprisinga γδ T cell activator and an interleukin-2 polypeptide, for separateuse, for regulating the activity of γδ T cells in a mammalian subject.

The invention further concerns the use of a γδ cell activator and aninterleukin-2 polypeptide, for the manufacture of a pharmaceuticalcomposition for regulating the activity of γδ T cells in a mammaliansubject, the γδ T cell activator and interleukin-2 polypeptide beingadministered separately to the subject. Preferably, said γδT activatoris a synthetic γδT activator. Preferably, said interleukin-2 polypeptideis administered at low doses. Preferably, said interleukin-2 polypeptideis administered over a period of time comprised between 1 and 10 days.More preferably, said interleukin-2 polypeptide is administered at adaily dose comprised between 0.2 and 2 MU per day, even more preferablybetween 0.2 and 1.5 MU, further preferably between 0.2 and 1 MU.Optionally, said daily dose of interleukin-2 polypeptide is administeredas a single injection or in two injections. Preferably, said γδ T cellactivator is administered as a single dose at the beginning of thetreatment. Optionally, said γδ T cell activator is a ligand of the Treceptor of γδ T lymphocytes. Preferably, said γδ T cell activator isadministered at least twice, more preferably with an interval of abouttwo weeks to about eight weeks between treatments, still more preferablywith an interval of about three to about four weeks between treatments.Optionally, said γδ T cell activator is administered is administered atleast three, four or six times. Preferably, said pharmaceuticcomposition increases the biological activity of γδ T cells in saidsubject. Preferably, said pharmaceutic preparation increases the numberof circulating γδ T cells in said subject. Preferably, said γδ T cellactivator is administered in an amount sufficient to expand the γδ Tcell population in a subject to reach between 30-90% of totalcirculating lymphocytes. Optionally, said γδ T cell activator isadministered in an amount sufficient to induce an at least 10-foldincrease in the γδ T cell population in a subject. Preferably, said γδcells are Vγ9/Vδ2 T cells. Optionally, said γδ T cell activator isadministered by intravenous infusion, preferably said infusion takesplace during about 5 to about 120 min, more preferably during about 5 toabout 30 min. Optionally, said subject is a human subject having acancer, an infectious disease, an auto-immune disease or an allergicdisease. In a particular embodiment, said pharmaceutical composition isused for treating cancer in a subject. Preferably, said cancer is asolid tumor. In a particular embodiment, said cancer is a solid tumorwith metastases. Alternatively, said cancer is a haematological tumor,preferably a lymphoma. Optionally, said cancer is a metastatic tumor.Preferably, said cancer is selected from the group consisting of lung,colorectal, prostate, breast or epidermoid head or neck tumors. In apreferred aspect of the invention, said cancer is a renal cancer,preferably a metastatic renal cancer. Alternatively, said cancer isselected from the group consisting of a melanoma, ovarian cancer,pancreas cancer, neuroblastoma, head or neck cancer, bladder cancer,renal cancer, brain cancer and gastric cancer. In an other particularembodiment, said pharmaceutical composition is used for treating aninfectious disease in a subject. In a further particular embodiment,said pharmaceutical composition is used for treating an autoimmunedisease in a subject. In an additional particular embodiment, saidpharmaceutical composition is used for treating a disease caused by orassociated with pathological cells sensitive to lysis by γδT cells in asubject.

The invention concerns a method of treating a cancer, an infectiousdisease, an autoimmune disease or an allergic disease in a subject,comprising separately administering to a subject in need thereof aneffective amount of a γδT activator and an interleukin-2 polypeptide.Preferably, said γδT activator is a synthetic γδT activator. Preferably,said interleukin-2 polypeptide is administered at low doses. Preferably,said interleukin-2 polypeptide is administered over a period of timecomprised between 1 and 10 days. More preferably, said interleukin-2polypeptide is administered at a daily dose comprised between 0.2 and 2MU per day, even more preferably between 0.2 and 1.5 MU, furtherpreferably between 0.2 and 1 MU. Optionally, said daily dose ofinterleukin-2 polypeptide is administered as a single injection or intwo injections. Preferably, said γδ cell activator is administered as asingle dose at the beginning of the treatment. Optionally, said γδ Tcell activator is a ligand of the T receptor of γδ T lymphocytes. In aparticular embodiment, said γδ T cell activator is a PED or PHD compoundand is administered as a single injection at a dose comprised between 10and 50 mg/kg, at the beginning of the treatment, and wherein saidinterleukin-2 polypeptide is administered at a daily dose comprisedbetween 0.2 and 2 MU per day over a period of time comprised between 1and 10 days. Preferably, said γδ T cell activator is administered atleast twice, more preferably with an interval of about two weeks toabout eight weeks between treatments, still more preferably with aninterval of about three to about four weeks between treatments.Optionally, said γδ T cell activator is administered is administered atleast three, four or six times. Preferably, said pharmaceuticcomposition increases the biological activity of γδ T cells in saidsubject. Preferably, said pharmaceutic preparation increases the numberof circulating γδ cells in said subject. Preferably, said γδ T cellactivator is administered in an amount sufficient to expand the γδ Tcell population in a subject to reach between 30-90% of totalcirculating lymphocytes. Optionally, said γδ T cell activator isadministered in an amount sufficient to induce an at least 10-foldincrease in the γδ cell population in a subject. Preferably, said γδ Tcells are Vγ9/Vδ2 T cells. Optionally, said γδ T cell activator isadministered by intravenous infusion, preferably said infusion takesplace during about 5 to about 120 min, more preferably during about 5 toabout 30 min. Preferably, said method is a method of treating a cancer.Preferably, said cancer is a solid tumor. In a particular embodiment,said cancer is a solid tumor with metastases. Alternatively, said canceris a haematological tumor, preferably a lymphoma. Optionally, saidcancer is a metastatic tumor. Preferably, said cancer is selected fromthe group consisting of lung, colorectal, prostate, breast or epidermoidhead or neck tumors. In a preferred aspect of the invention, said canceris a renal cancer, preferably a metastatic renal cancer. Alternatively,said cancer is selected from the group consisting of a melanoma, ovariancancer, pancreas cancer, neuroblastoma, head or neck cancer, bladdercancer, renal cancer, brain cancer and gastric cancer.

A number of suitable γδ T cell activators are provided herein that canbe used in accordance with any of the methods described herein.Preferably a γδ T cell activator is a compound capable of regulating theactivity of, or preferably of inducing the proliferation of a γδ cell ina pure population of γδ T cell clones, preferably Vγ9/Vδ2 T cells. Morepreferably, the γδ T cell activator is a compound capable of regulatingthe activity of a γδ T cell in a population of γδ T cell clones when theγδ T cell activator is present in culture at a concentration of lessthan 100 mM, less than 10 mM or most preferably less than 1 mM. Furtherpreferred γδ cell activators are described in detail herein.

Particularly preferred γδ T cell activators comprise a compositioncomprising a compound of formula (II):

in which X is an halogen (preferably selected from L Br and Cl), B is Oor NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+represents one (or several, identical or different) organic or mineralcation(s) (including the proton), and n is an integer from 2 to 20, A isO, NH, CHF, CF₂ or CH₂, and Y is O⁻Cat+, a nucleoside, or a radical-A-R, wherein R is selected from the group of 1), 2) or 3). Preferably,Y is O⁻Cat+, or a nucleoside. More preferably, Y is O⁻Cat+. Preferably,R1 is a methyl. Preferably, A is O or CH₂. More preferably, A is O.Preferably, n is 2. Preferably, X is a bromide. Preferably, B is O.Preferably, m is 1 or 2. More preferably, m is 1. Preferably such acompound is administered in a dose to humans that is between about 10mg/kg and about 100 mg/kg, preferably between about 5 mg/kg and about 60mg/kg, together with a pharmaceutically acceptable carrier. In otherpreferred aspects such compound is administered in two or moretreatments; preferably the compound is administered in a dose to humansthat is calculated according to the formula (A) single dose (mg/kg)=(10to y)*N (A) where N is the number of weeks between treatments and y is I00; more preferably in a dose to humans that is calculated according tothe formula B single dose (mg/kg)=(5 to 60)*N (B); or preferably in adose of about 20 mg/kg. Yet more preferably, said γδT activator isadministered by intravenous infusion in a dose to humans that iscalculated according to the formula (C) single dose (mg/kg)=(10 to100)*N (C) where N is the number of weeks between treatments such that Nis between about 3 and about 4.

Other particularly preferred γδ T cell activators include a compositioncomprising a compound of formula (XII):

in which R₃, R₄, and R₅ , identical or different, are a hydrogen or(C₁-C₃)alkyl group, W is —CH— or —N—, R₆ is an (C₂-C₃)acyl, an aldehyde,an (C₁-C₃)alcohol, or an (C₂-C₃)ester Cat+ represents one (or severalidentical or different) organic or mineral cation(s) (including theproton), B is O or NH, m is an integer from 1 to 3, A is O, NH, CHF, CF₂or CH₂, and Y is O⁻Cat+, a nucleoside, or a radical -A-R, wherein R isselected from the group of 1), 2) or 3). Preferably, Y is O⁻Cat+, or anucleoside. More preferably, Y is O⁻Cat+. Preferably, A is O or CH₂.More preferably, A is O. More preferably, R₃ and R₅ are a methyl and R₄is a hydrogen. More preferably, R₆ is —CH₂—OH, —CHO, —CO—CH₃ or—CO—OCH₃. Preferably, B is O. Preferably, m is 1 or 2. More preferably,m is 1. Optionally, the double-bond between W and C is in conformationtrans (E) or cis (Z). More preferably, the double-bond between W and Cis in conformation trans (E). Preferably such a compound is administeredin a dose to humans that is between about 10 μg/kg to 20 mg/kg, morepreferably between 10 μg/kg to 5 mg/kg or yet more preferably between 10μg/kg to 1 mg/kg, together with a pharmaceutically acceptable carrier.In other preferred aspects such compound is administered in two or moretreatments; preferably the compound is administered in a dose to humansthat is calculated according to the formula (A) single dose(mg/kg)=(0.001 to y)*N (A) where N is the number of weeks betweentreatments and y is 100; preferably in a dose to humans that iscalculated according to the formula B single dose (mg/kg)=(0.01 to 20)*N(B); preferably in in a dose to humans that is calculated according tothe formula C single dose (mg/kg)=(0.01 to 5)*N (C); preferably in adose to humans that is calculated according to the formula D single dose(mg/kg)=(0.02 to 5)*N (D); or preferably in a dose of about 0.5 mg/kg.Most preferably said γδT activator is administered by intravenousinfusion in a dose to humans that is calculated according to the formula(E) single dose (mg/kg)=(0.01 to 20)*N (E) where N is the number ofweeks between treatments such that N is between about 3 and about 4.

In preferred aspects of any of the methods described herein, the γδTactivator is a synthetic γδT activator.

In other preferred aspects of any of the methods described herein, theγδ T cell activator is selected from phosphohalohydrin (PHD) compounds,phosphoepoxyde (RED) compounds, and alkylamines. More preferably, the γδT cell activator is selected from the following compounds:

-   3-(bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP)-   3-(iodomethyl)-3-butanol-1-yl-diphosphate (IHPP)-   3-chloromethyl)-3-butanol-1-yl-diphosphate (ClHPP)-   3-bromomethyl)3-butanol-1-yl-triphosphate (BrHPPP)-   3-iodomethyl)-3-butanol-1-yl-triphosphate (IHPPP)-   α,γ-di-[3-bromomethyl)-3-butanol-1-yl]-triphosphate (diBrHTP)-   α,γ-di-[3-(iodomethyl)-3-butanol-1-yl]-triphosphate (diIHTP)-   3,4,-epoxy-3-methyl-1-butyl-diphosphate (Epox-PP)-   3,4,epoxy-3-methyl-1-butyl-triphosphate (Epox-PPP)-   α,γ-di-3,4,epoxy-3-methyl-1-butyl-triphosphate (di-Epox-TP)

In other aspects, the s T cell activator is a PED or PHD compound and isadministered as a single injection at a dose comprised between 10 and 50mg/kg, at the beginning of the treatment, and wherein the interleukin-2polypeptide is administered at a daily dose comprised between 0.2 and 2MU per day over a period of time comprised between 1 and 10 days.

In further aspects, the invention provides a method for stimulating a γδT cell in a warm-blooded animal, said method comprising administering,in more than one treatment, a composition comprising a compound offormula XII, together with a pharmaceutically acceptable carrier, to awarm-blooded animal, wherein the γδ T cell activator is administered inmore than one treatment with an interval of about two weeks to abouteight weeks between treatments.

In further aspects, the invention provides a method for stimulating a γδT cell in a warm-blooded animal said method comprising administering, inmore than one treatment, a composition comprising a compound of formulaII, together with a pharmaceutically acceptable carrier, to awarm-blooded animal, wherein the γδ T cell activator is administered inmore than one treatment with an interval of about two weeks to abouteight weeks between treatments.

In another aspect of the invention, disclosed is a method forstimulating a γδ T cell in a human subject, said method comprisingadministering a composition comprising a HDMAPP compound in a dose thatis between about 10 μg/kg and about 2.5 mg/kg, together with apharmaceutically acceptable carrier, to the subject. In further aspects,the invention provides a method for stimulating a γδ T cell in a humansubject, said method comprising administering a composition comprising aCHDMAPP compound in a dose that is between about 10 μg/kg and about 2.5mg/kg, together with a pharmaceutically acceptable carrier, to thesubject.

In further aspects, the invention provides a method for stimulating a γδcell in a human subject, said method comprising administering acomposition comprising a CBrHPP compound in a dose that is between about5 mg/kg and about 60 mg/kg, together with a pharmaceutically acceptablecarrier, to the subject.

The present invention also provides method for administering a γδ T cellactivator to a subject. Thus, in any methods of the invention, saidmethods may comprise administering the γδ T cell activator byintravenous infusion. Preferably, each infusion takes place during about5 to about 120 min, or more preferably during about 5 to about 30 min.Most preferably, only a single such infusion (also referred to as singleshot) of the γδ T cell activator takes place on any given day (e.g.within a period of less than 24 hours). Preferably only a singleinfusion takes place in an administration of a γδ T cell activator, thatis, per treatment cycle.

In one particularly preferred aspect, the invention relates to a methodof regulating γδ T cells in a human subject, the method comprising:administering, preferably by intravenously infusing, a compositioncomprising a compound of formula XII into said subject a dose of between10 μg/kg to 20 mg/kg, more preferably between 10 μg/kg to 5 mg/kg or yetmore preferably between 10 μg/kg to 1 mg/kg, of said compound perkilogram of the subject's weight, preferably in a single administration(single shot), and preferably when by infusion within a period of lessthan 24 hours. In one particularly preferred aspect, the inventionrelates to a method of regulating Vγ9/Vδ2⁺ T cells in a human subject,the method comprising: administering, preferably by intravenouslyinfusing, a composition comprising a HDMAPP or CHDMAPP compound (offormula XV and XVI respectively) into said subject a dose of between 10μg/kg to 20 mg/kg, more preferably between 10 μg/kg to 5 mg/kg or yetmore preferably between 10 μg/kg to 1 mg/kg, of said compound perkilogram of the subject's weight, preferably in a single administration(single shot), and preferably when by infusion within a period of lessthan 24 hours.

In yet further aspects, the invention provides a pharmaceuticalcomposition containing a therapeutically effective amount of CHDMAPP asan active ingredient, together with a pharmaceutically acceptablecarrier. Also provided is a pharmaceutical composition containing atherapeutically effective amount of CBrHPP as an active ingredient,together with a pharmaceutically acceptable carrier.

DESCRIPTION OF THE DRAWINGS

FIG. 1

Successive intravenous injections of increasing doses of BrHPP do notsustain γδ cell amplification in the periphery of M. fascicularis. Twoanimals received successively 1, 4, 16 and 32 mg/kg BrHPP daily andtheir PBMCs were monitored by flow cytometry. Total blood was stainedwith anti-delta2-FITC and anti-CD3-PE antibodies until 20 days afterBrHPP administration.

FIG. 2

A—BrHPP and IL2 co-administration induces reproducible γδ cell increasein M. fascicularis. Two animals received 20 mg/kg BrHPP (“1 dose”) andtwo others received 4 mg/kg BrHPP daily during 5 days (“split doses”).All of them were co-injected with 0.9 million UI IL2 per day for 5 days.The four BrHPP/IL2 co-treated animals showed an increase in peripheralγδ cells, with a peak at day 7. As a control, a fifth animal was treatedwith IL2 alone and exhibited no change in its peripheral γδ rate. ABrHPP/IL2 co-administration to this animal at day 14 demonstrated it wasable to answer with the same increase in γδ rate 7 days after.

B—Flow cytometry analyses of total blood of M. fascicularis at day 7after administration. These graphs represent theanti-delta2-FITC/anti-CD3-PE staining on total blood of the animalstreated with IL2 alone (Z604 D7 IL2 only) or with BrHPP and IL2 (Z604,X973, Z059, Z135, Z714, D7 IL2+BrHPP) 7 days after the administration.

C—Absolute number of circulating γδ cells increases 5 to 40-fold inBrHPP/IL2 co-injected M. fascicularis. Absolute numbers of peripheral γδcells were determined by flow cytometry in the four BrHPP/IL2co-injected animals. Peripheral y3 cell number was found 5 to 40 timeshigher at day 7 after administration than before, depending on animals.A mean 24-fold increase was obtained for a total amount of 20 mg/kgBrHPP injected, single doses being more efficient than split ones.

FIG. 3

A Peripheral γδ cell rate increase upon BrHPP/IL2 administration isdose-dependant. Upper panel: A first injection was done with increasingamounts (0, 0.2,4, 20, 80 mg/kg) of BrHPP in 10 animals (numbered 2031to 2040), with 2 animals per dose (1 male+1 female). Lower panel: Sameanimals underwent a second injection 3 weeks after the first injectionwith even higher amounts of BrHPP (20, 80, 120, 160 mg/kg). Animals thatreceived the lowest doses at first injection (groups 0.2 and 4 mg/kg)were injected with the two new highest doses (120 and 160 mg/kg) tominimize potential effects of first injection on second injection.

B—Circulating to cell number increase upon BrHPP/IL2 co-treatment isalso dose-dependant. The fold increase in peripheral γδ cell number wascalculated as the ratio of γδ absolute count 7 days after treatment onγδ absolute count before treatment. Despite some discrepancy betweenanimals in the same dose-group, this increase is clearly dose-dependant.

C—Flow cytometry images of total blood of animal #2034, before and 5days after it received 160 mg/kg BrHPP, stained with anti-gamma9-FITCand anti-CD3-PE antibodies.

FIG. 4

A—Primate γδ cells respond in vivo equally to the first and the secondBrHPP/IL2 co-treatment. The mean amplification in γδ cell numberobserved 7 days after the first 20 mg/kg BrHPP treatment (fractionatedor not) is comparable to the mean increase observed after the 20 mg/kgBrHPP recall.

B—γδ amplification in vivo decreases after successive BrHPP/IL2administration. In this experiment, two animals received successively 80mg/kg and 22 days after 20 mg/kg, or 20 mg/kg followed by 80 mg/kg. Ineach case, the response in γδ cell number fold increase is lower at thefirst recall of a given dose than at first injection (left panel).Moreover, the five females were treated with a second and a third recallat 80 mg/kg, at 3 weeks intervals. The resulting amplification rates isstill detectable but become lower at each recall (right panel).

FIG. 5

A—Serum TNFα is produced in vivo after BrHPP/IL2 co-treatment. TNFα wasdetected by Elisa in the serum of the monkeys first treated with EL2 andincreasing doses of BrHPP (0 to 80 mg/kg, with two animals per dose, CfFIG. 3A), 1 and 4 hours after BrHPP injection.

B—In vivo peripheral BrHPP-amplified primate γδ cells produce cytokinesin response to BrHPP challenge. As a fourth BrHPP/EL2 treatment, females2032 and 2034 received 80 mg/kg BrHPP, which induced 3- and 7.9-foldincreases in γδ cell number respectively. At the time of γδ cellperipheral peak (day 5 after the 4^(th) injection), these animals werere-injected with 80 mg/kg BrHPP (without IL2) and serum INFγ and TNFαwere detected 60 and 120 minutes after injection by Elisa.

FIG. 6

Different IL2 co-treatment do not influence the maintenance ofperipheral γδ rate.

Concomitantly with 20 mg/kg BrHPP, animals received subcutaneously thefollowing IL2 treatment: 0.15 million units twice daily for 9 days(animal Z059), 0.3 million units twice daily for 5 days (Z135), 0.9million units twice daily for 5 days (animal Z714) or 9 days (animalX973).

FIG. 7

Primate γδ cell number fold increase in response to increasing doses ofPhosphostim.

The first and second administrations of Phosphostim and IL-2 resulted ina clear dose-related elevation of peripheral Vγ9Vδ2 T cells at Day 7 asdetermined by flow cytometry, which is represented in FIG. 7.

FIG. 8

Serum cytokine (INFγ and TNFα) production in Phosphostim treatedprimates.

The effects of Phosphostim treatment on the production of cytokines(INFγ and TNFα) production in the serum was studied twice:

-   -   after the first infusion, in all treated animals: a significant        production of systemic TNFα (serum concentrations around 60 and        120 pg/ml) was evidenced in both animals having received 80        mg/kg, 1 hour after BrHPP injection;    -   in two females (F2032 & F2034), which received 80 mg/kg (without        IL-2) during the peak (Day 7) of the 4^(th) injection.

Serum TNFα and INFγ concentration evolutions for both animals are shown,demonstrating that Vγ9Vδ2 T cells amplified in vivo upon BrHPP/IL-2co-treatment also produce detectable amounts of systemic cytokines.

FIG. 9

In vitro cytotoxicity of expanded Vγ9Vδ2 T cells from mRCC patients.

Lytic activities of BrHPP-amplified γδ T cells were measured againstclassical control targets (Raji and Daudi), primary autologous normaland tumor cell lines of the selected patients in a 4 h standard ⁵¹Crrelease assay for the five patients.

FIGS. 10 to 13

In vivo (monkey) dose ranging studies with HDMAPP and BrhPP

The dose-range effect of HDMAPP and BrHPP in vivo as determined bydetermining numbers of γδ T cells by flow cytometry are shown in FIGS.10 to 13.

FIGS. 10A and 10B show the absolute cell count (/mm³ blood) for HDMAPPand BrHPP respectively.

FIGS. 11A and 11B show the percentage γδ cells of total circulatinglymphocytes for HDMAPP and BrHPP respectively.

FIGS. 12A and 12B show the fold γδ T cell increase in absolute cellcount (/mm³ blood) for HDMAPP and BrHPP respectively.

FIGS. 13A and 13B show the fold increase in percentage of totalcirculating lymphocytes for HDMAPP and BrHPP respectively.

FIG. 14

A. Comparison of BrHPP and HDMAPP in vitro activities FIG. 14A shows thein vitro EC50 for the compounds BrHPP, HDMAPP and theaminobisphosphonate compound Zoledronate®. The in vitro biologicalactivity of γδ T cell amplification from human PBMCs (in the presence ofrhIL2) was assessed using a TNFα release assay.

B. Comparison of BrHPP and HDMAPP in vivo activities FIG. 14B shows thein vivo EC50 for the compounds: for BrHPP the EC50 is about 1 nM whilefor HDMAPP the EC50 is about 5 pM. By contrast, the less potentZoledronate® showed an EC50 value of about 1 μM. The biological activityof γδ T cell amplification from human PBMCs was assessed by counting γδcell using flow cytometry.

FIGS. 15 to 19

In vivo efficacy of human γδ T cells Nod-Scid/Tumor Model

Tumor growth in the first few days after administration of human PBMLand BrHPP treatment to a Nod-Scid mouse is shown in FIG. 15.

Phenotyping and homing of human cells: In the peritoneal cavity: theweekly check of the IP phenotype of treated mice showed a human γδ cellpresence only in the BrHPP treated mice. The relative numbers of γδ Tcells in the blood is shown in FIG. 16 and in the peritoneal cavity inFIG. 17. Human γδ T cells represent a higher percentage of the human CD3T cells. In the mice recipient organs: phenotyping is carried out atsacrifice (4 weeks after the PBMC and BrHPP treated or not treatedgroups); the major human cells present in those organs are human CD3+ Tcells with 99% expression of the Ab TcR However in the tumor, human γδcells were present only in the BrHPP treated group.

While tumor size increased in the first few days after treatment, tumorsize decreased thereafter quickly in the PBMC/BrHPP and IL2 treatedgroup. FIG. 19 shows that from day 7 onwards the tumor size shrank. Inthe PBMC group, after short arrest, the size grows and no significantdifference was observed between the tumors sizes in this group and thoseof the negative control.

DETAILED DESCRIPTION

Definitions

Within the context of the present invention, the expression “regulatingthe activity of γδ T cells” designates causing or favoring an increasein the number and/or biological activity of such cells in a subject.Regulating thus includes without limitation modulating (e.g.,stimulating) expansion of such cells in a subject and/or, for instance,triggering of cytokine secretion (e.g., TNFα or IFNγ). As indicated, γδT cells normally represent between about 1-10% of total circulatinglymphocytes in a healthy adult human subject. The present invention canbe used to significantly increase the γδ T cells population in asubject, particularly to reach 30-90% of total circulating lymphocytes,typically 40-90%, more preferably from 50-90%. In typical embodiments,the invention allows the selective expansion of γδ cells in a subject,to reach 60-90% of total circulating lymphocytes, preferably 70-90%,more preferably from 80-90%. Regulating also includes, in addition or inthe alternative, modulating the biological activity of γδ cells in asubject, particularly their cytolytic activity or theircytokine-secretion activity. The invention defines novel conditions andstrategies for increasing the biological activity of γδ cells towardstarget cells.

As used herein, the term “EC50” with respect to regulating the activityof γδ T cells, refers to the efficient concentration of the subjectcompositions which produces 50% of its maximum response or effect withrespect to such activity of γδ T cells.

As used herein, the term “EC100” with respect to regulating the activityof γδ T cells, refers to the efficient concentration of the subjectcompositions which produces its maximum response or effect with respectto such activity of γδ T cells.

Where hereinbefore and hereinafter numerical terms are used, they aremeant to include the numbers representing the upper and lower limits.For example, “between 1 and 3” stands for a range “from and including 1up to and including 3”, and “in the range from 1 to 3” would stand for“from and including 1 up to and including 3”. The same is true whereinstead of numbers (e.g. 3) words denoting numbers are used (e.g.“three”).

Where “comprising” is used, this can preferably be replaced by“consisting essentially of”, more preferably by “consisting of”.

Where “about” is used in connection with a number, this preferably meansthe number ±5%, more preferably the number plus 5%, most preferably thenumber itself without “about”. For example, “about 100” would stand for“from and including 85 to and including 115”. Where “about” is used inconnection with numeric ranges, for example “about 1 to about 3”, or“between about one and about three”, preferably the definition of“about” given for a number in the last sentence is applied to eachnumber defining the start and the end of a range separately. Preferably,where “about” is used in connection with any numerical values, the“about” can be deleted.

“Weekly” stands for “about once a week” (meaning that more than onetreatment is made with an interval of about one week betweentreatments), the about here preferably meaning ±1 day (that is,translating into “every 6 to 8 days”); most preferably, “weekly standsfor “once every 7 days”.

“3-weekly” or “three-weekly” stands for “about once every three weeks”(meaning that more than one treatment is made with an interval of aboutthree weeks between treatments), the about here preferably meaning ±3days (that is, translating into every 18 to 24 days); most preferably,“weekly” stands for “once every 21 days” (=every third week).

Whenever within this whole specification “treatment of a proliferativedisease” or “treatment of a tumor”, or “treatment of cancer” or the likeis mentioned with reference to γδ T cell activator(s), there is meant:

-   a) a method of treatment (=for treating) of a proliferative disease,    said method comprising the step of administering (for at least one    treatment) a γδ cell activator, (preferably in a pharmaceutically    acceptable carrier material) to a warm-blooded animal, especially a    human, in need of such treatment, in a dose that allows for the    treatment of said disease (=a therapeutically effective amount),    preferably in a dose (amount) as specified to be preferred    hereinabove and hereinbelow;-   b) the use of a γδ T cell activator for the treatment of a    proliferative disease; or a γδ T cell activators, for use in said    treatment (especially in a human);-   c) the use of a γδ T cell activators, for the manufacture of a    pharmaceutical preparation for the treatment of a proliferative    disease; and/or-   d) a pharmaceutical preparation comprising a dose of a γδ T cell    activator that is appropriate for the treatment of a proliferative    disease; or any combination of a), b), c) and d), in accordance with    the subject matter allowable for patenting in a country where this    application is filed;-   e) a method of using a γδ T cell activator for the manufacture of a    pharmaceutical preparation for the treatment of a proliferative    disease, comprising admixing said γδ T cell activator with a    pharmaceutically acceptable carrier. In cases where a tumor disease    or a specific tumor (e.g. colon tumor, colon carcinoma or colon    cancer; or prostate tumor, prostate carcinoma or prostate cancer)    are mentioned instead of “proliferative disease”, categories a)    to e) are also encompassed, meaning that the respective tumor    disease can be filled in under a) to e) above instead of    “proliferative disease”, in accordance with the patentable subject    matter.

As further described herein, several γδ T cell activators have beenfound to regulate the activity of γδ T cells in vitro with at the nano-and pico-molar concentration level. Based on in vivo studies with firstand second γδT activators, BrHPP and HDMAPP, it has now been found thatthese compounds are able to induce increases in biological activity aswell as high rates of proliferation of γδ T cells in vivo in a primate.This enables a new strategy of stimulation of γδ T cells in vivo.

It has now also been found that a strategy of stimulation of γδ T cellscan be effective in humans for the treatment of tumors, including solidtumors, and furthermore also in solid tumors with metastases. A γδ Tcell activating compound, BrHPP (also referred to as Phosphostim), wasused in a clinical study to demonstrate that treatment of humans havingmetastatic renal carcinoma by introducing ex vivo activated γδ cellscould result in the effective treatment of solid tumors. Briefly, BrHPPwas used in an adoptive autologous cellular immunotherapy processdesigned to produce large numbers of highly enriched Vγ9Vδ2 T cells frompatient's cytapheresis. Ex vivo stimulation allows the generation ofcritical numbers of effector cells for therapeutic purposes. TheBrHPP-stimulated γδ T cells were obtained by a 2-week manufacturingprocess. The initial cell preparation consists of PBMCs from blood fromeither fresh or frozen cytapheresis. The cells are expanded for twoweeks in a closed system, with sequential addition of defined dosageIL-2 to the culture medium after a unique GMP-grade Phosphostimstimulation. The manufacturing process is much simpler than most currentcellular therapy approaches using conventional CD8+ T cell lines orclones: there is no final separation or purification step nor use offeeder cells; the specific TCR-mediated signal provided by Phosphostimis sufficient to trigger the IL-2-dependent expansion of the Vγ9Vδ2subset, which becomes dominant in the culture. Several doses of the γδcellular product can be manufactured from one frozen cytapheresis.Typically, 100 million frozen PBMCs from cytapheresis yield 2 to 5billions cells. The BrHPP-stimulated γδ T cells are currently being usedin a Phase I clinical trial in metastatic Renal Cell Carcinoma (mRCC).The trial is currently on going at the second dose level of 4 billionscells after achieving correct tolerance of the first 1 billion celldose. Preliminary results from the ongoing trial are promising, as theevaluable patients showed signs of anti-tumor activity in the form ofstable disease.

In was also found that autologous γδ T cells could lyse, as a functionof the effector to target cell ratio, tumors cells obtained from humanpatients having metastatic renal cell carcinoma, while not substantiallycausing lysis of normal (non-tumor) cells from the same patient. Asdescribed herein, BrHPP is a synthetic phosphoantigen that specificallyexpands Vγ9Vδ2 T cells from healthy donors' Peripheral Blood MononuclearCells (PBMC). These expanded Vγ9Vδ2 T cells lysed lymphoma targets andsome established Renal Carcinoma Cell (RCC) lines.

In the present study, primary normal and tumor renal cells from RCCpatients were established, and the effect of the BrHPP on PBMCs of thesepatients was investigated. This permitted assessment of the lyticpotential of expanded Vγ9Vδ2 T cells against primary normal and tumorrenal cells in an is autologous setting. This cytotoxic activity wascompared with other autologous effectors cells, like LAK cells(Lymphokine-Activated Killer cells) for example.

The inventors performed a series of experiments suggesting that thecompound BrHPP could also be used to treat solid tumors in vivo, andmoreover metastatic tumors. In one set of experiments the inventors useda NOD/SCID mouse model of cancer to which human γδ T cells wereintroduced to demonstrate that a γδ T cell activator could prevent orarrest growth of metastatic renal cell tumor cells in culture.

The inventors further elucidated the pharmacokinetics of γδ T cells inprimates in a series of experiments using several different γδ T cellactivators. Provided are the findings that γδ cells stimulated in aprimate reach their peak numbers after between about 5 and 7 daysfollowing administration of the γδ cell activator. Furthermore, resultsshowed that γδ T cell activators can be administered repeatedly andre-stimulate γδ cell activity in vivo. However, re-stimulation of γδ Tcells is optimally not performed prior to peak expansion of y9 T, andmoreover that such re-stimulation be performed once the γδ T cellpopulation has returned to substantially the rate prior to the firststimulation. Furthermore, each administration of a compound to stimulateγδ T cells is optimally performed by a single shot, for example byinfusion in the examples presented herein. In one example comparing twoγδ cell activators BrHPP and HDMAPP, dosages providing optimal γδ T cellbiological activity and proliferation increases were determined. Indetermining the EC50 of respective compounds, the comparison revealed acorrespondence of in vitro and in vivo EC50 dosages. Interestingly,assessment of the activity of the HDMAPP compound revealed that incomparison with its previously reported activity, low doseadministration regimens for this compound may be used, as was confirmedin the EC50 in primates.

Thus, in a first aspect, the present invention relates to the treatmentof a tumor, preferably a solid tumor, wherein a γδ cell activator isadministered to a warm-blooded animal, especially a human, preferably ahuman in need of such treatment, especially in a therapeuticallyeffective amount.

A variety of cancers and other proliferative diseases including, but notlimited to, the following can be treated using the methods andcompositions of the invention:

-   -   carcinoma, including that of the bladder, breast, colon, kidney,        liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin,        including squamous cell carcinoma;    -   hematopoietic tumors of lymphoid lineage, including leukemia,        acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell        lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins        lymphoma, hairy cell lymphoma and Burketts lymphoma;    -   hematopoietic tumors of myeloid lineage, including acute and        chronic myelogenous leukemias and promyelocytic leukemia;    -   tumors of mesenchymal origin, including fibrosarcoma and        rhabdomyoscarcoma;    -   other tumors, including melanoma, seminoma, teratocarcinoma,        neuroblastoma and glioma;    -   tumors of the central and peripheral nervous system, including        astrocytoma, neuroblastoma, glioma, and schwannomas;    -   tumors of mesenchymal origin, including fibrosarcoma,        rhabdomyoscaroma, and osteosarcoma; and    -   other tumors, including melanoma, xeroderma pigmentosum,        keratoacanthoma, seminoma, thyroid follicular cancer and        teratocarcinoma.

As discussed, the methods of the invention may also be used for thetreatment or prevention of an autoimmune disease or an infectiousdisease.

Where hereinbefore and subsequently a tumor, a tumor disease, acarcinoma or a cancer are mentioned, also metastasis in the originalorgan or tissue and/or in any other location are implied alternativelyor in addition, whatever the location of the tumor and/or metastasis is.

In a preferred aspect, the γδ T cell activator may increase thebiological activity of γδ T cells, preferably increasing the activationof γδ cells, particularly increasing cytokine secretion from γδ cells orincreasing the cytolytic activity of γδ cells, with or without alsostimulating the expansion of γδ T cells. Thus in one aspect, the presentinvention relates to methods for the treatment of a disease, especiallya proliferative disease, and more preferably a solid tumor, particularlya solid tumor having metastases, where a γδ cell activator comprising acompound of the formula I, especially a γδ T cell activator according toformulas I to XVII, especially γδ T cell activator selected from thegroup consisting of BrHPP, CBrHPP, HDMAPP HDMAPP and epoxPP, isadministered in an amount and under conditions sufficient to increasethe activity γδ T cells in a subject, preferably in an amount and underconditions sufficient to increase cytokine secretion by γδ T cellsand/or to increase the cytolytic activity of γδ cells. In typicalembodiments, a γδ T cell activator allows the cytokine secretion by γδ Tcells to be increased at least 2, 3, 4, 10, 50, 100-fold, as determinedin vitro.

Cytokine secretion and cytolytic activity can be assessed using anyappropriate in vitro assay, or those provided in the examples herein.For example, cytokine secretion can be determined according to themethods described in Espinosa et al. (J. Biol. Chem., 2001, Vol. 276,Issue 21, 18337-18344), describing measurement of TNF-α release in abioassay using TNF-α-sensitive cells. Briefly, 10⁴ γδT cells/well wereincubated with stimulus plus 25 units of IL2/well in 100 μl of culturemedium during 24 h at 37° C. Then, 50 μl of supernatant were added to 50μl of WEHI cells plated at 3×10⁴ cells/well in culture medium plusactinomycin D (2 μg/ml) and LiCl (40 mM) and incubated for 20 h at 37°C. Viability of the TNF-α-sensitive cells and measured with a3-(4,5-dimethylthiazol-2-yl}2,5-diphenyltetrazolium bromide assay. 50 μlof 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma;2.5 mg/ml in phosphate-buffered saline) per well were added, and after 4h of incubation at 37° C., 50 μl of solubilization buffer (20% SDS, 66%dimethyl formamide, pH 4.7) were added, and absorbance (570 nm) wasmeasured. Levels of TNF-α release were then calculated from a standardcurve obtained using purified human rTNF-α (PeproTech, Inc., Rocky Hill,N.J.). Interferon-γ released by activated T cells was measured by asandwich enzyme-linked immunosorbent assay. 5×10⁴ γδT cells/well wereincubated with stimulus plus 25 units of IL2/well in 100 [2l of culturemedium during 24 h at 37° C. Then, 50 μl of supernatant were harvestedfor enzyme-linked immunosorbent assay using mouse monoclonal antibodies(BIOSOURCE. Camarillo, Calif.).

A preferred assay for cytolytic activity is a ⁵¹Cr release assay. Inexemplary assays, the cytolytic activity of γδ T cells is measuredagainst autologous normal and tumor target cell lines, or controlsensitive target cell lines such as Daudi and control resistant targetcell line such as Raji in 4 h ⁵¹Cr release assay. In a specific example,target cells were used in amounts of 2×10³ cells/well and labeled with100 μCi ⁵¹Cr for 60 minutes. Effector/Target ( E/T) ratio ranged from30:1 to 3.75:1. Specific lysis (expressed as percentage) is calculatedusing the standard formula [(experimental-spontaneousrelease/total-spontaneous release)×100].

In another aspect, the present invention relates to methods for thetreatment of a disease, especially a proliferative disease, and morepreferably a solid tumor, particularly a solid tumor having metastases,where a γδ T cell activator, especially a γδ T cell activator accordingto formulas I to XVII, especially γδ T cell activator selected from thegroup consisting of BrHPP, CBrHPP, HDMAPP HDMAPP and epoxPP, isadministered in an amount and under conditions sufficient to stimulatethe expansion of the γδ cell population in a subject, particularly toreach 30-90% of total circulating lymphocytes, typically 40-90%, morepreferably from 50-90%. In typical embodiments, the invention allows theselective expansion of γδ cells in a subject, to reach 60-90% of totalcirculating lymphocytes, preferably 70-90%, more preferably from 80-90%.Percentage of total circulating lymphocytes can be determined accordingto methods known in the art. A preferred method for determining thepercentage of γδ T cells in total circulating lymphocytes is by flowcytometry, examples of appropriate protocols described in the examplesherein.

In another embodiment, the present invention relates to methods for thetreatment of a disease, especially a proliferative disease, and morepreferably a solid tumor, particularly a solid tumor having metastases,where a γδ T cell activator, especially a γδ T cell activator accordingto formulas I to XVI, especially γδ T cell activator selected from thegroup consisting of BrHPP, CBrHPP, HDMAPP HDMAPP and epoxPP, isadministered in an amount and under conditions sufficient to stimulatethe expansion of the γδ T cell population in a subject, particularly toincrease by more than 2-fold the number of γδ T cells in a subject,typically at least 10-fold, more preferably at least 20-fold. In typicalembodiments, the invention allows the selective expansion of γδ T cellsin a subject, to increase by at least 2, 4, 10, 20, or 50-fold thenumber of γδ T cells in a subject, more preferably at least 100 or200-fold. The number of γδ T cells in a subject is preferably assessedby obtaining a blood sample from a patient before and afteradministration of said γδ T cell activator and determining thedifference in number of γδ T cells present in the sample. A preferredmethod for determining the number of γδ T cells by flow cytometry,examples of appropriate protocols described in the examples herein.

In another aspect, the present invention relates to methods for thetreatment of a disease, especially a proliferative disease, and morepreferably a solid tumor, particularly a solid tumor having metastases,where a γδ T cell activator, especially a γδ T cell activator accordingto formulas I to XVI, especially γδ T cell activator selected from thegroup consisting of BrBPP, CBrEPP, HDMAPP HDMAPP and epoxPP, isadministered in an amount and under conditions sufficient to stimulatethe expansion of the γδ T cell population in a subject, particularly toreach a circulating γδ T cell count of at least 500 γδ T cells/mm3 in asubject, typically at least 1000 γδ T cells/mm 3, more preferably atleast 2000 γδ T cells/mm3. The circulating γδ T cell count in a subjectis preferably assessed by obtaining a blood sample from a patient beforeand after administration of said γδ T cell activator and determining thenumber of γδ T cells in a given volume of sample. A preferred method fordetermining the number of γδ T cells by flow cytometry, examples ofappropriate protocols described in the examples herein.

In a further aspect, the present invention relates to an in vivo regimenfor the treatment of a proliferative disease, especially a solid tumorand more particularly a solid tumor having metastases, where a γδ T cellactivator, especially a γδ T cell activator according to formulas I toXVI, especially γδ T cell activator selected from the group consistingof BrHPP, CBrHPP, HDMAPP HDMAPP and epoxPP, is administered to awarm-blooded animal, especially a human, in a dose that is higher(preferably at least 10%, 20%, 30% higher) than the singleadministration Efficient Concentration value giving half of the maximumneffect (EC50) of γδ T cell biological activity or population expansion,or more preferably a dose that is at least 50%, or more preferably atleast 60%, 75%, 85% or preferably between about 50% and 100% of thesingle administration Efficient Concentration value giving the maximumeffect.

In preferred aspects, one or more (preferably at least two) furtherdoses preferably each within the dose range mentioned herein areadministered in one or preferably more than one further treatmentcycle(s), especially with an interval between the treatment cycles ofmore than one week or preferably more than two weeks after the precedingtreatment, more preferably after about two to about eight (8) weeks,most preferably about three (3) to about four (4) weeks after thepreceding treatment, respectively. Generally, this treatment regimenwhere a dose is administered in two or more treatment cycles withperiods of time between one to eight, preferably three to four weeks oftime between administrations is preferred over more frequent treatmentswith lower doses, lower increases in γδ T cell biological activity orlower expansion of γδ T cell population.

Preferably, dosage (single administration) of a compound of formula Ifor treatment is between about 1 μg/kg and about 1.2 g/kg.

It will be appreciated that the above dosages related to a group ofcompounds, and that each particular compound may vary in optimal doses,as further described herein for exemplary compounds. Nevertheless,compounds are preferably administered in a dose sufficient tosignificantly increase the biological activity of γδ T cells or tosignificantly increase the γδ T cell population in a subject. Said doseis preferably administered to the human by intravenous (i.v.)administration during 2 to 180 min, preferably 2 to 120 min, morepreferably during about 5 to about 60 min, or most preferably duringabout 30 min or during about 60 min.

In preferred exemplary compounds, a compound of formula II to XI isadministered in a dosage (single administration) between about 0.1 mg/kgand about 1.2 g/kg, preferably between about 10 mg/kg and about 1.2g/kg, more preferably between about 5 mg/kg and about 100 mg/kg, evenmore preferably between about 5 μg/kg and 60 mg/kg. Most preferably,dosage (single administration) for three-weekly or four-weekly treatment(treatment every three weeks or every third week) is between about 0.1mg/kg and about 1.2 g/kg, preferably between about 10 mg/kg and about1.2 g/kg, more preferably between about 5 mg/kg and about 100 mg/kg,even more preferably between about 5 μg/kg and 60 mg/kg. This dose ispreferably administered to the human by intravenous (i.v.)administration during 2 to 180 min, preferably 2 to 120 min, morepreferably during about 5 to about 60 min, or most preferably duringabout 30 min or during about 60 min.

In preferred exemplary compounds, a compound of formula XII to XVII, isadministered in a dosage (single administration) between about 1 μg/kgand about 100 mg/kg, preferably between about 10 μg/kg and about 20mg/kg, more preferably between about 20 μg/kg and about 5 mg/kg, evenmore preferably between about 20 μg/kg and 2.5 mg/kg. Most preferably,dosage (single administration) for three-weekly or four-weekly treatment(treatment every three weeks or every third week) is between about 1μg/kg and about 100 mg/kg, preferably between about 10 μg/kg and about20 mg/kg, more preferably between about 20 μg/kg and about 5 mg/kg, evenmore preferably between about 20 μg/kg and 2.5 mg/kg. This dose ispreferably administered to the human by intravenous (i.v.)administration during 2 to 180 min, preferably 2 to 120 min, morepreferably during about 5 to about 60 min, or most preferably duringabout 30 min or during about 60 min.

On other aspects, the dosage of a γδ T cell activator may be determinedas a function of its maximal tolerated dose or highest tested dose innon-human animals. The present invention thus discloses an in vivoregimen for the treatment of a proliferative disease, especially a solidtumor and more particularly a solid tumor having metastases, where a γδT cell activator, especially a γδ T cell activator according to formulaL especially γδ T cell activator selected from the group consisting ofBrHPP, CBrHPP, HDMAPP HDMAPP and epoxPP, is administered in a dose thatis between about 1 and about 100%, preferably between about 25 and 100%,of the (single administration) maximal tolerated dose (MTD) to awarm-blooded animal, especially a human.

Treatment Cycles

In further methods, the inventors have devised administration regimensproviding improved regulation of γδ T cell activity based on the in vivokinetics of γδ T cell regulating pyrophosphate compounds.

The invention provides a method of regulating the activity of γδ T cellsin a mammalian subject, the method comprising administering to a subjectin need thereof an effective amount of a γδ T cell activator accordingto a treatment cycle in which γδ T cell activity, preferably the γδ Tcell rate (number of γδ T cells), is allowed to return to substantiallybasal rate prior to a second administration of the compound. As furtherdescribed herein, in preferred embodiments, at least about one week, butmore preferably at least about two weeks, are required for a patient'sγδ T cell rate to return to substantially basal rate.

As further shown in the examples, the inventors have found that cyclesshorter than about 7 days do not permit suitable stimulation of γδ Tcell activity. The course of a preferred cycle is an at least 1-weeklycycle, but more preferably at least a 2-weekly cycle (at least about 14days), or more preferably at least 3-weekly or 4weekly, though cyclesanywhere between 2-weekly and 4weekly are preferred. Also effective andcontemplated are cycles of up to 8-weekly, for example 5-weekly,6-weekly, 7-weekly or 8-weekly.

In one preferred embodiment, administration of the γδ T cell activatoroccurs on the first day of a 2-weekly to 4-weekly cycle (that is, anabout 14 to 28 day weeks repeating cycle). In a preferred embodiment,the γδ T cell activator is administered only the first day of the2-weekly to 4-weekly, or preferably 3 weekly, cycle.

In preferred embodiments, administration of the γδ T cell activatoroccurs on the first day of a 1-weekly to 4-weekly cycle. In a preferredembodiment, the γδ cell activator is administered only on the first dayof the 1-weekly to 4-weekly cycle. In a preferred embodiment, the γδ Tcell activator is administered only on the first day of the 1-weekly to4-weekly cycle.

In particularly preferred embodiments, administration of the γδ T cellactivator occurs on the first day of a 3-weekly to 4-weekly cycle. In apreferred embodiment, the γδ T cell activator is administered only onthe first day of the 3-weekly to 4-weekly cycle. In a preferredembodiment, the γδ T cell activator is administered only on the firstday of the 3-weekly to 4-weekly cycle.

As mentioned, a subject will preferably be treated for at least twocycles, or more preferably for at least three cycles. In other aspect,treatment may continue for a greater number of cycles, for example atleast 4, 5, 6 or more cycles can be envisioned. At the end of eachcycle, the cycle of dosing may be repeated for as long as clinicallytolerated and the tumor is under control or until tumor regression.Tumor “control” is a well recognized clinical parameter, as definedabove. In a preferred embodiment, the cycle of dosing is repeated for upto about eight cycles

Co-Treatment with Cytokine

In other embodiments, the methods of the invention comprises furtheradministering a cytokine. While the compounds of the invention may beused with or without further administration, in a preferred aspect acytokine can be administered, wherein said cytokine is capable ofincreasing the expansion of a γδ T cell population treated with a γδ Tcell activator compound, preferably wherein the cytokine is capable ofinducing an expansion of a γδ T cell population which is greater thanthe expansion resulting from administration of the γδ T cell activatorcompound in the absence of said cytokine. A preferred cytokine is aninterleukin-2 polypeptide.

A cytokine having γδ T cell proliferation inducing activity, mostpreferably the interleukin-2 polypeptide, is administered at low doses,typically over a period of time comprised between 1 and 10 days. The γδT cell activator is preferably administered in a single dose, andtypically at the beginning of a cycle.

In preferred aspects, a cytokine, most preferably IL-2, is administereddaily for up to about 10 days, preferably for a period of between about3 and 10 days, or most preferably for about 7 days. Preferably, theadministration of the cytokine begins on the same day (e.g. within 24hours of) as administration of the γδ T cell activator. It will beappreciated that the cytokine can be administered in any suitable schemewithin said regimen of between about 3 and 10 days. For example, in oneaspect the cytokine is administered each day, while in other aspects thecytokine need not be administered on each day. When the cytokine isadministered for about 7 to about 14 days, a 4-weekly treatment cycle ispreferred. When the first component is administered for about 4 days, a3-weekly day treatment cycle is preferred.

The invention thus also relates to the use of a synthetic γδ T cellactivator and a cytokine. In most preferred embodiments, the cytokine isan interleukin-2 polypeptide. The cytokine can be used for themanufacture of a pharmaceutical composition for regulating the activityof γδ T cells in a mammalian subject. More specifically, the γδ T cellactivator and interleukin-2 polypeptide are administered separately.Even more preferably, the interleukin-2 polypeptide is administered atlow doses, typically over a period of time comprised between 1 and 10days. In a preferred embodiment, the γδ T cell activator is administeredin a single dose, typically at the beginning of the treatment.

The present invention more specifically relates to the use of a γδ Tcell activator and an interleukin-2 polypeptide, for the manufacture ofa pharmaceutical composition for treating cancer in a subject, whereinsaid γδ T cell activator and interleukin-2 polypeptide are administeredseparately to the subject. More preferably, the interleukin-2polypeptide is administered at low doses, typically over a period oftime comprised between 1 and 10 days, and/or the γδ T cell activator isadministered in a single dose at the beginning of the treatment.

In an other specific embodiment, the present invention relates to theuse of a γδ T cell activator and an interleukin-2 polypeptide, for themanufacture of a pharmaceutical composition for treating an infectiousdisease in a subject, wherein said γδ T cell activator and interleukin-2polypeptide are administered separately to the subject. More preferably,the interleukin-2 polypeptide is administered at low doses, typicallyover a period of time comprised between 1 and 10 days, and/or the γδ Tcell activator is administered in a single dose at the beginning of thetreatment.

In a further embodiment, the present invention relates to the use of aγδ T cell activator and an interleukin-2 polypeptide, for themanufacture of a pharmaceutical composition for treating an autoimmunedisease in a subject, wherein said γδ T cell activator and interleukin-2polypeptide are administered separately to the subject. More preferably,the interleukin-2 polypeptide is administered at low doses, typicallyover a period of time comprised between 1 and 10 days, and/or the γδ Tcell activator is administered in a single dose at the beginning of thetreatment.

The invention also relates to methods of treating a cancer, aninfectious disease, an autoimmune disease or an allergic disease in asubject, comprising separately administering to a subject in needthereof an effective amount of a γδ T cell activator and aninterleukin-2 polypeptide.

The above methods and treatments may be used alone or in combinationwith other active agents or treatments. For instance, for the treatmentof tumors, the invention may be used in combination with otheranti-tumor agents or treatments, such as chemotherapy, radiotherapy orgene therapy.

The invention also relates to a product comprising a γδ T cell activatorand an interleukin-2 polypeptide, for separate use, for regulating theactivity of γδ T cells in a mammalian subject.

In preferred embodiments, the cytokine is administered during the firstcycle of treatment with the γδT activator, and during each subsequentcycle of treatment with the γδT activator. However, it will beappreciated that the treatment regimen of the invention may be modifiedsuch that the cytokine is not administered in the first cycle oftreatment. That is, the method may comprise:

-   -   (a) providing to a subject at least one first cycle of        treatment, said first cycle comprising administering a synthetic        γδT activator on the first day of a 1-weekly to 4-weekly cycle,        or more preferably a 2-weekly to 4-weekly cycle, wherein no        cytokine is administered during said first cycle;    -   (b) providing at least a second cycle of treatment, said second        cycle comprising administering a synthetic γδT activator on the        first day of a 1-weekly to 4-weekly cycle, or more preferably a        2-weekly to 4-weekly cycle, and administering a cytokine for a        period of between 1 and about 10 days; and    -   (c) optionally repeating step (b) for any suitable number of        further cycles.

In other aspects, it will be appreciated that the treatment regimen ofthe invention may be modified such that the cytokine is not administeredin one or more of the subsequent cycles of treatment.

That is, the method comprises:

-   -   (a) providing to a subject at least one first cycle of        treatment, said first cycle comprising administering a synthetic        γδT activator on the first day of a 1-weekly to 4weekly cycle,        or more preferably a 2-weekly to 4weekly cycle, and        administering a cytokine for a period of between 1 and about 10        days;    -   (b) providing at least a second cycle of treatment, said second        cycle comprising administering a synthetic γδT activator on the        first day of a 1-weekly to 4-weekly cycle, or more preferably a        2-weekly to 4weekly cycle, wherein no cytokine is administered        during said second cycle; and    -   (c) optionally repeating steps (a) or (b) for any suitable        number of further cycles.        Mode of Use

As disclosed herein, the γδ T cell activator is preferably administeredas a single shot. When used in a treatment comprising more than oneadministration, the γδ T cell activator is preferably administered as asingle shot at the beginning of a treatment cycle. As shown in theexperimental section, such administration schedule provides a remarkableincrease in the activity of γδ T cells in a subject. The activeingredients may be administered through different routes, typically byinjection or oral administration. Injection may be carried out intovarious tissues, such as by intravenous, intra-peritoneal,intra-arterial, intramuscular, intra-dermic, subcutaneous, etc.

Most preferably, the γδ T cell activator is administered by intravenous(i.v.) administration. Preferably said infusion is during 2 to 180 min,preferably 2 to 120 min, more preferably during about 5 to about 30 min,most preferably during about 10 to about 30 min, e.g. during about 30min. As further described herein, the invention discloses that a briefstimulation of γδ T cell activity is sufficient to achieve the γδ T cellregulating effect. Thus, preferably where a γδ T cell activatingcompound has a short serum half-life, for example having a serumhalf-life of less than about 48 hours, less than about 24 hours, or lessthan about 12 hours, a rapid infusion is used. Said rapid infusion ispreferably between about 10 minutes and 60 minutes, or more preferablyabout 30 minutes.

When an administration regimen comprises both aγδ T cell activator andan interleukin-2 polypeptide, said compounds are preferably separatelyadministered Within the context of the present invention, the term“separately administered” indicates that the active ingredients areadministered at a different site or through a different route or througha different schedule to the subject. Accordingly, the ingredients aregenerally not mixed together prior to administration, although they maybe combined in a unique package in suitable separated containers.

In a preferred embodiment, the active ingredients are administeredthrough different schedules: the synthetic γδ T cell activator isadministered as a single shot, at the beginning of the treatment, andthe interleukin-2 polypeptide is administered over a prolonged period oftime, typically between 1 and 10 days. As shown in the experimentalsection, such administration schedule provides a remarkable increase inthe activity of γδ T cells in a subject.

The active ingredients may be administered through different routes,typically by injection or oral administration. Injection may be carriedout into various tissues, such as by intravenous, intra-peritoneal,intra-arterial, intramuscular, intra-dermic, subcutaneous, etc.Preferred administration routes for the synthetic activators areintravenous and intramuscular. Preferred administration routes for thecytokine are subcutaneous, intravenous and intramuscular.

A specific embodiment of the present invention relates to the use of (i)a synthetic γδT activator selected from a PED or a PHD compound and (ii)an interleukin-2 polypeptide, for the manufacture of a pharmaceuticalcomposition for treating cancer, an infectious disease or an auto-immunedisease in a subject, wherein said synthetic γδT activator andinterleukin-2 polypeptide are administered separately to the subject.More preferably, the interleukin-2 polypeptide is administered at lowdoses, typically over a period of time comprised between 1 and 10 days,and/or the synthetic γδT activator is administered in a single dose atthe beginning of the treatment, preferably at a dose comprised between0.5 and 80 mg/kg, more preferably between 1 and 60, even more preferablybetween 2 and 60.

Dosage of γδ T Cell Activators

As discussed, specific dosage ranges suitable for the administration ofγδ T cell activators to increase the activity of γδ T cells aredisclosed herein. Nevertheless, it will be appreciated that the dose ofactivator may be adapted by the skilled artisan, depending on the natureof the activator, its specific-activity, EC50, stability and/orpharmacokinetics, as well as on the type of subject, etc. Severalmethods for doing so with respect to γδ T cell activators are alsoprovided herein. Preferably, the γδ T cell activator is administered ina human subject at a dose comprised between 0.01 and 200 mg/kg,preferably between 0.05 and 80 mg/kg, more preferably between 0.1 and60, even more preferably between 1 and 60 mg/kg. The activator may beadministered as a single dose, at the beginning of the treatment, ordistributed over several days. Unexpectedly, however, the inventionshows that a significantly higher effect is obtained when the activatoris administered in a single dose at the beginning of the treatment.Typical dosages are comprised between 1 and 60 mg/kg, more preferablybetween 1 and 50 mg/kg. Appropriate dosages may be deduced fromexperiments conducted in animals, by normalizing with respect to themean body weight and mean body surface. For instance, it can becalculated that efficient doses of between 2 and 300 mg/kg in acynomolgus monkey (having a mean body weight of about 3 kg and mean bodysurface of about 0.3 m²) correspond to an efficient dosage of between0.5 and 80 mg/kg in a human subject (having a mean body weight of about65 kg and mean body surface of about 1.8 m²). It should be understoodthat different dosages may be used, including higher dosages,considering the low toxicity observed in vivo with the syntheticactivators.

The invention further provides method which may be used to determine theappropriate dosage ranges for further γδ T cell activators based ontheir in vitro activities. In one aspect, the invention provides in vivoand in vitro dose-effect curves for two γδ T cell activators showingcorrespondence in in vitro and in vivo γδ T cell stimulating activities,particularly correspondence of EC50 values. The invention thus alsoprovides methods of determining the dosage of a γδ T cell activator, aswell as dosages for administration to human subjects for said testcompounds based on the examples provided herein. An appropriate dosagerange for a compound can be determined by (a) determining the in vitroγδT activating potency of a compound, preferably using an assay methodas described in the examples herein, and (b) comparing said activity ofthe test compound to the in vitro activity obtained using a compound forwhich the in vivo activity is known, preferably a BrHPP or HDMAPPcompound, and computing an in vivo dosage or dosage range proportionalto that obtained with the compound for which the in vivo activity isknown. Preferably determining the in vitro activity comprisesdetermining the EC50. In this way, a dosage range can be determined, forexample useful for selecting a starting dose for studies in non-humanmammalian subjects, preferably cynomolgus monkeys. Further precision canbe obtained by administering different doses within the range tosubjects and assessing γδ T cell activity, for examples using the assaysdescribed herein.

The present invention also provides that a brief stimulation of γδ Tcell activity is sufficient to stimulate an increase in γδ T cellactivity. Thus, suitable γδ T cell activating compound include compoundshaving short as well as longer half-lives. In one aspect, a γδ T cellactivator having a short serum half-life, for example having a serumhalf-life of less than about 48 hours, less than about 24 hours, or lessthan about 12 hours, is administered in a dose that is higher(preferably at least 110%, 120%, 130%, or 150% of the EC50) than thesingle administration Efficient Concentration value giving half of themaximum effect (EC50) of γδ T cell biological activity or populationexpansion, or more preferably a dose that is at least 50%, or morepreferably at least 60%, 75%, 85% or preferably between about 50% and100% of the single administration Efficient Concentration value givingthe maximum effect (EC100). Preferably said administration is byintravenous infusion, during between about 10 minutes and about 60minutes.

In other aspect, appropriate dosage may be determined as a function ofthe Maximal Tolerated Dose (MTD). The MTD is determined according tostandard procedures; preferably, in warm-blooded animals the MTD in caseof oral or intravenous administration is determined as the dose of asingle administration where no death occurs and a loss of body weight ofless than 40, preferably less than 25, percent (%) is found in thetreated warm-blooded animal individual (this term here mainly referringto an animal; for humans see below). In other aspects, where dose rangestudies in animal have not demonstrated an MTD, the highest tested dosemay be considered in place of the MTD.

The MTD may vary depending on the population of the patients which maybe defined by tumor type, age range, gender, tumor stage and the like.While in animals, the most preferable way of determining the MTD can beanalogous to that shown in the Examples presented below, in humans theMTD may generally be determined by starting with one singleadministration of a very low dose, e.g. 1/10th of the LD10 (i.e., thedose that is lethal to 10% of animals) in the most sensitive animalspecies in which toxicology studies have been performed. Dose escalationfor the next dose level is 100%, unless grade 2 toxicity is seenaccording to the US National Cancer Institute Revised Common ToxicityCriteria, in which case dose escalation will be 67%. Dose escalation forsubsequent dose levels is in the range of 25% to 67%. For example, threepatients are usually treated at one dose level and observed for acutetoxicity for one course of treatment before any more patients areentered. If none of the three patients experience DLT (dose-limitingtoxicity), then the next cohort of three patients is treated with thenext higher dose. If two or more of the three patients experience DLT,then three more patients are treated at the next lower dose unless sixpatients have already been treated at that dose. If one of threepatients treated at a dose experiences DLT, then three more patients aretreated at the same level. If the incidence of DLT among those patientsis one in six, then the next cohort is treated at the next higher dose.In general, if two or more of the six patients treated at a dose levelexperience DLT, then the MTD is considered to have been exceeded, andthree more patients are treated at the next lower dose as describedabove. The MTD is defined as the highest dose studied for which theincidence of DLT was less than 33%. Usually dose escalation forsubsequent courses in the same patient—i.e. intrapatient doseescalation—is not permitted. Alternatively, dose steps may be defined bya modified Fibonacci series in which the increments of dose forsucceeding levels beyond the starting dose are 100%, 67%, 50% and 40%,followed by 33% for all subsequent levels. Finally, the MTD may be foundby methods described in Simon, R., et al., J. Nat. Cancer Inst. 89(15),1997, p. 1138-1147.

The DLT generally includes (but is not limited to) any drug-relateddeath and most drug-related grade 3 and 4 toxicities, including febrileneutropenia (see also US National Cancer Institute Revised CommonToxicity Criteria). See especially the examples.

In the above methods and uses, the subject is preferably a humansubject, such as a subject having a cancer, an infectious disease, anautoimmune disease or an allergic disease. The invention is indeedsuitable to treat all conditions caused by or associated with thepresence of pathological cells which are sensitive to γδ T cell lysis.

The invention is particularly suited to stimulate the anti-tumorimmunity of a subject having a solid or hematopoietic tumor, such as alymphoma, bladder cancer, multiple myeloma, renal cell carcinoma, etc.

Nevertheless, the invention is also suitable to stimulate an anti-viralimmune response in a subject having an infection by a virus selectedfrom HIV, CMV, EBV, Influenza virus, HCV, HBV, etc.

The invention is also suitable to stimulate an immune response in asubject having an infection by a pathogen causing tuberculosis, malaria,tularemia, colibacillosis, etc.

The invention is also suitable to treat (e.g., to stimulate an immuneresponse in) a subject having an autoimmune disease, such as diabetes,multiple sclerosis, rheumatoid arthritis, etc. or a subject having anallergic disease, including asthma, airway hyper-responsiveness, etc.

Unless otherwise indicated, the dosages for administration to a warmblooded animal, particularly humans provided herein are indicated inpure form (anionic form) of the respective compound. Purity level forthe active ingredient depending on the synthesis batch can be used toadjust the dosage from actual to anionic form and vice-versa.

Synthetic γδ T Lymphocyte Activators

An advantageous aspect of this invention resides in the use of asynthetic γδT lymphocytes activating compound. Indeed, the inventionshows that a potent and targeted expansion and activation of γδT cellscan be obtained in vivo by trigerring one single metabolic pathway,using defined activating compounds following a particular administrationschedule.

The term “synthetic γδT lymphocyte activating compound”, and the term“synthetic γδT cell activating compound” used interchangeable,designates a molecule artificially produced, which can activate γδTlymphocytes. More particularly, the term synthetic γδT lymphocyteactivating compound designates a molecule produced ex vivo or in vitro.It is more preferably a ligand of the T receptor of γδT lymphocytes. Theactivator may by of various nature, such as a peptide, lipid, smallmolecule, etc. It may be a purified or otherwise artificially produced(e.g., by chemical synthesis, or by microbiological process) endogenousligand, or a fragment or derivative thereof or an antibody havingsubstantially the same antigenic specificity. The activator is mostpreferably a synthetic chemical compound capable of selectivelyactivating Vγ9Vδ2 T lymphocytes. Selective activation of Vγ9Vδ2 Tlymphocytes indicates that the compound has a selective action towardsspecific cell populations, and essentially does not activate other Tcell sub-types, such as Vδ1 T cells. Such selectivity, as disclosed inthe present application, suggests that preferred compounds can cause aselective or targeted activation of the proliferation or biologicalactivity of Vγ9Vδ2 T lymphocytes.

Preferably a synthetic γδ T lymphocyte activator is a compound capableof regulating the activity of a γδ T cell in a population of γδ T cellclones in culture. The synthetic γδ T lymphocyte is capable ofregulating the activity of a γδ T cell population of γδ T cell clones ina at millimolar concentration, preferably when the γδ T cell activatoris present in culture at a concentration of less than 100 mM. Optionallya synthetic γδ T lymphocyte is capable of regulating the activity of aγδ T cell in a population of γδ T cell clones at millimolarconcentration, preferably when the γδ cell activator is present inculture at a concentration of less than 10 mM, or more preferably lessthan 1 mM. Regulating the activity of a γδ T cell can be assessed by anysuitable means, preferably by assessing cytokine secretion, mostpreferably TNF-α secretion as described herein. Methods for obtaining apopulation of pure γδ T cell clones is described in Davodeau et al,(1993) and Moreau et al, (1986), the disclosures of which areincorporated herein by reference. Preferably the compound is capable ofcausing at least a 20%, 50% or greater increase in the number of γδ Tcells in culture, or more preferably at least a 2-fold increase in thenumber of γδ T cells in culture.

Synthetic γδ T lymphocyte activators comprise the compounds of formula(I):

wherein Cat+ represents one (or several, identical or different) organicor mineral cation(s) (including proton);

-   m is an integer from 1 to 3;-   B is O, NH, or any group capable to be hydrolyzed;-   Y═O⁻Cat+, a C₁-C₃ alkyl group, a group -A-R, or a radical selected    from the group consisting of a nucleoside, an oligonucleotide, a    nucleic acid, an amino acid, a peptide, a protein, a monosaccharide,    an oligosaccharide, a polysaccharide, a fatty acid, a simple lipid,    a complex lipid, a folic acid, a tetrahydrofolic acid, a phosphoric    acid, an inositol, a vitamin, a co enzyme, a flavonoid, an aldehyde,    an epoxyde and a halohydrin;-   A is O, NH, CHF, CF₂ or CH₂; and,-   R is a linear, branched, or cyclic, aromatic or not, saturated or    unsaturated, C₁-C₅₀ hydrocarbon group, optionally interrupted by at    least one heteroatom, wherein said hydrocarbon group comprises an    alkyl, an alkylenyl, or an alkynyl, preferably an alkyl or an    alkylene, which can be substituted by one or several substituents    selected from the group consisting of: an alkyl, an alkylenyl, an    alkynyl an epoxyalkyl, an aryl an heterocycle, an alkoxy, an acyl,    an alcohol, a carboxylic group (—COOH), an ester, an amine, an amino    group (—NH₂), an amide (—CONH₂), an imine, a nitrile, an hydroxyl    (—OH), a aldehyde group (—CHO), an halogen, an halogenoalkyl, a    thiol (—SH), a thioalkyl, a sulfone, a sulfoxide, and a combination    thereof.

In a particular embodiment, the substituents as defined above aresubstituted by at least one of the substituents as specified above.

Preferably, the substituents are selected from the group consisting of:an (C₁-C₆)alkyl, an (C₂-C₆)alkylenyl, an (C₂-C₆)alkynyl, an(C₂-C₆)epoxyalkyl, an aryl, an heterocycle, an (C₁-C₆)alkoxy, an(C₂-C₆)acyl, an (C₁-C₆)alcohol, a carboxylic group (—COOH), an(C₂-C₆)ester, an (C₁-C₆)amine, an amino group (—NH₂), an amide (—CONH₂),an (C₁-C₆)imine, a nitrile, an hydroxyl (—OH), a aldehyde group (—CHO),an halogen, an (C₁-C₆)halogenoalkyl, a thiol (—SH), a (C₁-C₆)thioalkyl,a (C₁-C₆)sulfone, a (C₁-C₆)sulfoxide, and a combination thereof.

More preferably, the substituents are selected from the group consistingof: an (C₁-C₆)alkyl, an (C₂-C₆)epoxyalkyl an (C₂-C₆)alkylenyl an(C₁-C₆)alkoxy, an (C₂-C₆)acyl, an (C₁-C₆)alcohol, an (C₂-C₆)ester, an(C₁-C₆)amine, an (C₁-C₆)imine, an hydroxyl a aldehyde group, an halogen,an (C₁-C₆)halogenoalkyl, and a combination thereof.

Still more preferably, the substituents are selected from the groupconsisting of : an (C₃-C₆)epoxyalkyl, an (C₁-C₃)alkoxy, an (C₂-C₃)acylan (C₁-C₃)alcohol, an (C₂-C₃)ester, an (C₁-C₃)amine, an (C₁-C₃)imine, anhydroxyl, an halogen, an (C₁-C₃)halogenoalkyl, and a combinationthereof. and a combination thereof. Preferably, R is a(C₃-C₂₅)hydrocarbon group, more preferably a (C₅-C₁₀)hydrocarbon group.

In the context of the present invention, the term “alkyl” morespecifically means a group such as methyl ethyl, propyl isopropyl,butyl, isobutyl, tert-butyl pentyl hexyl, heptyl, octyl, nonyl decyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl nonadecyl, eicosyl, heneicosyl, docosyl and theother isomeric forms thereof. (C₁-C₆)alkyl more specifically meansmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,hexyl and the other isomeric forms thereof. (C₁-C₃)alkyl morespecifically means methyl, ethyl, propyl, or isopropyl.

The term “alkenyl” refers to an alkyl group defined hereinabove havingat least one unsaturated ethylene bond and the term “alkynyl” refers toan alkyl group defined hereinabove having at least one unsaturatedacetylene bond. (C₂-C₆)alkylene includes a ethenyl, a propenyl(1-propenyl or 2-propenyl), a 1- or 2-methylpropenyl, a butenyl(1-butenyl, 2-butenyl, or 3-butenyl), a methylbutenyl, a2-ethylpropenyl, a pentenyl (1-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl), an hexenyl (1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl), and the other isomeric forms thereof (C₂-C₆)alkynyl includesethynyl 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,1-pentynyl 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 4-hexynyl, or 5-hexynyl and the other isomeric forms thereof.

The term “epoxyalkyl” refers to an alkyl group defined hereinabovehaving an epoxide group. More particularly, (C₂-C₆)epoxyalkyl includesepoxyethyl, epoxypropyl, epoxybutyl, epoxypentyl, epoxyhexyl and theother isomeric forms thereof. (C₂-C₃)epoxyalkyl includes epoxyethyl andepoxypropyl.

The “aryl” groups are mono-, bi- or tri-cyclic aromatic hydrocarbonshaving from 6 to 18 carbon atoms. Examples include a phenyl, α-naphthyl,β-naphthyl or anthracenyl group, in particular.

“Heterocycle” groups are groups containing 5 to 18 rings comprising oneor more heteroatoms, preferably 1 to 5 endocyclic heteroatoms. They maybe mono-, bi- or tricyclic. They may be aromatic or not. Preferably, andmore specifically for R₅, they are aromatic heterocycles. Examples ofaromatic heterocycles include pyridine, pyridazine, pyrimidine,pyrazine, furan, thiophene, pyrrole, oxazole, thiazole, isothiazole,imidazole, pyrazole, oxadiazole, triazole, thiadiazole and triazinegroups. Examples of bicycles include in particular quinoline,isoquinoline and quinazoline groups (for two 6-membered rings) andindole, benzimidazole, benzoxazole, benzothiazole and indazole (for a6-membered ring and a 5-membered ring). Nonaromatic heterocyclescomprise in particular piperazine, piperidine, etc.

“Alkoxy” groups correspond to the alkyl groups defined hereinabovebonded to the molecule by an —)— (ether) bond. (C₁-C₆)alkoxy includesmethoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy and the otherisomeric forms thereof (C₁-C3)alkoxy includes methoxy, ethoxy,propyloxy, and isopropyloxy.

“Alcyl” groups correspond to the alkyl groups defined hereinabove bondedto the molecule by an —CO— (carbonyl) group. (C₂-C₆)acyl includesacetyl, propylacyl, butylacyl, pentylacyl, hexylacyl and the otherisomeric forms thereof. (C₂-C₃)acyl includes acetyl, propylacyl andisopropylacyl.

“Alcohol” groups correspond to the alkyl groups defined hereinabovecontaining at least one hydroxyl group. Alcohol can be primary,secondary or tertiary. (C₁-C₆)alcohol includes methanol, ethanol,propanol, butanol, pentanol, hexanol and the other isomeric formsthereof. (C₁-C₃)alcohol includes methanol, ethanol, propanol andisopropanol.

“Ester” groups correspond to the alkyl groups defined hereinabove bondedto the molecule by an —COO— (ester) bond. (C₂-C₆)ester includesmethylester, ethylester, propylester, butylester, pentylester and theother isomeric forms thereof (C₂-C₃)ester includes methylester andethylester.

“Amine” groups correspond to the alkyl groups defined hereinabove bondedto the molecule by an —N— (amine) bond. (C₁-C₆)amine includesmethylamine, ethylamine, propylamine, butylamine, pentylamine,hexylamine and the other isomeric forms thereof (C₁-C₃)amine includesmethylamine, ethylamine, and propylamine.

“Imine” groups correspond to the alkyl groups defined hereinabove havinga (—C═N—) bond. (C₁-C₆)imine includes methylimine, ethylimine,propylimine, butylimine, pentylimine, hexylimine and the other isomericforms thereof. (C₁-C₃)imine includes methylimine, ethylimine, andpropylimine.

The halogen can be Cl, Br, I, or F, more preferably Br or F.

“Halogenoalkyl” groups correspond to the alkyl groups definedhereinabove having at least one halogen. The groups can bemonohalogenated or polyhalogenated containing the same or differenthalogen atoms. For example, the group can-be an trifluoroalkyl (CF₃—R).(C₁-C₆)halogenoalkyl includes halogenomethyl, halogenoethyl,halogenopropyl, halogenobutyl, halogenopentyl, halogenohexyl and theother isomeric forms thereof. (C₁-C₃)halogenoalkyl includeshalogenomethyl, halogenoethyl, and halogenopropyl.

“Thioalkyl” groups correspond to the alkyl groups defined hereinabovebonded to the molecule by an —S— (thioether) bond. (C₁-C₆)thioalkylincludes thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl,thiohexyl and the other isomeric forms thereof. (C₁-C₃)thioalkylincludes thiomethyl, thioethyl, and thiopropyl.

“Sulfone” groups correspond to the alkyl groups defined hereinabovebonded to the molecule by an —SOO— (sulfone) bond. (C₁-C₆)sulfoneincludes methylsulfone, ethylsulfone, propylsulfone, butylsulfone,pentylsulfone, hexylsulfone and the other isomeric forms thereof.(C₁-C₃)sulfone includes methylsulfone, ethylsulfone and propylsulfone.

“Sulfoxyde” groups correspond to the alkyl groups defined hereinabovebonded to the molecule by an —SO— (sulfoxide) group. (C₁-C₆)sulfoxideincludes methylsulfoxide, ethylsulfoxide, propylsulfoxide,butylsulfoxide, pentylsulfoxide, hexylsulfoxide and the other isomericforms thereof. (C₁-C₃)sulfoxide includes methylsulfoxide,ethylsulfoxide, propylsulfoxide and 1 5 isopropylsulfoxide.

“Heteroatom” denotes N, S, or O.

“Nucleoside” includes adenosine, thymine, uridine, cytidine andguanosine.

In a particular embodiment, the hydrocarbon group is a cycloalkylenylsuch as a cyclopentadiene or a phenyl, or an heterocycle such as afuran, a pyrrole, a thiophene, a thiazole, an imidazole, a triazole, apyridine, a pyrimidine, a pyrane, or a pyrazine. Preferably, thecycloalkylenyl or the heterocycle is selected from the group consistingof a cyclopentadiene, a pyrrole or an imidazole. In a preferredembodiment, the cycloalkylenyl or the heterocycle is sustituted by analcohol. Preferably, said alcohol is a (C₁-C₃)alcohol.

In an other embodiment, the hydrocarbon group is an alkylenyl with oneor several double bonds. Preferably, the alkylenyl group has one doublebond. Preferably, the alkylenyl group is a (C₃-C₁₀)alkylenyl group, morepreferably a (C₄-C₇)alkylenyl group. Preferably, said alkylenyl group issubstituted by at least one functional group. More preferably, thefunctional group is selected from the group consisting of an hydroxy, an(C₁-C₃)alkoxy, an aldehyde, an (C₂-C₃)acyl, or an (C₂-C₃)ester. In amore preferred embodiment, the hydrocarbon group is butenyl substitutedby a group —CH₂OH. Optionally, said alkenyl group can be the isoformtrans (E) or cis (Z), more preferably a trans isoform (E). In a mostpreferred embodiment, the alkylenyl group is the(E)hydroxy-3-methyl-2-butenyl. In an other preferred embodiment, thealkylenyl group group is an isopentenyl, an dimethylallyl or anhydroxydimethylallyl.

In an additional embodiment, the hydrocarbon group is an alkyl groupsubstituted by an acyl. More preferably, the hydrocarbon group is an(C₄-C₇)alkyl group substituted by an (C₁-C₃)acyl.

In a further preferred embodiment, R is selected from the groupconsisting of:

wherein n is an integer from 2 to 20, R₁ is a (C₁-C₃)alkyl group, and R₂is an halogenated (C₁-C₃)alkyl, a (C₁-C₃)alkoxy-(C₁-C₃)alkyl, anhalogenated (C₂-C₃)acyl or a (C₁-C₃)alkoxy-(C₂-C₃)acyl. Preferably, R₁is a methyl or ethyl group, and R₂ is an halogenated methyl (—CH₂—X, Xbeing an halogen), an halogenated (C₂-C₃)acetyl, or(C₁-C₃)alkoxy-acetyl. The halogenated methyl or acetyl can be mono-,di-, or tri-halogenated. Preferably, n is an integer from 2 to 10, orfrom 2 to 5. In a more preferred embodiment, n is 2. In a most preferredembodiment, n is 2, R₁ is a methyl and R₂ is an halogenated methyl, morepreferably a monohalogenated methyl, still more preferably a bromidemethyl. In a particularly preferred embodiment, n is 2, R₁ is a methyl,R₂ is a methyl bromide. In a most preferred embodiment, R is3-(bromomethyl)-3-butanol-1-yl.

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group.Preferably, n is an integer from 2 to 10, or from 2 to 5. In a morepreferred embodiment, n is 2 and R1 is a methyl.

wherein R₃, R₄, and R₅ , identical or different, are a hydrogen or(C₁l₃)alkyl group, W is —CH— or —N—, and R₆ is an (C₂-C₃)acyl, analdehyde, an (C₁-C₃)alcohol, or an (C₂-C₃)ester. More preferably, R₃ andR₅ are a methyl and R₄ is a hydrogen. More preferably, R₆ is —CH₂—OH,—CHO, '1CO—CH₃ or —CO—OCH₃. Optionally, the double-bond between W and Cis in conformation trans (E) or cis (Z). More preferably, thedouble-bond between W and C is in conformation trans (E).

The group Y can allow to design a prodrug. Therefore, Y is enzymolabilegroup which can be cleaved in particular regions of the subject. Thegroup Y can also be targeting group. In a preferred embodiment, Y isO⁻Cat+, a group -A-R, or a radical selected from the group consisting ofa nucleoside, a monosaccharide, an epoxyde and a halohydrin. Preferably,Y is an enzymolabile group. Preferably, Y is O⁻Cat+, a group -A-R, or anucleoside. In a first preferred embodiment, Y is O⁻Cat+. In a secondpreferred embodiment, Y is a nucleoside.

In a preferred embodiment, Cat⁺ is H⁺, Na⁺, NH₄ ⁺, K⁺, Li⁺,(CH₃CH₂)₃NH⁺.

In a preferred embodiment, A is O, CHF, CF₂ or CH₂. More preferably, Ais O or CH₂.

In a preferred embodiment, B is O or NH. More preferably, B is O.

In a preferred embodiment, m is 1 or 2. More preferably, m is 1.

In one particular embodiment, synthetic γδT lymphocyte activatorscomprise the compounds of formula (II):

in which X is an halogen (preferably selected from I, Br and Cl), B is 0or NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+represents one (or several, identical or different) organic or mineralcation(s) (including the proton), and n is an integer from 2 to 20, A isO, NH, CHF, CF₂ or CH₂, and Y is O⁻Cat+, a nucleoside, or a radical-A-R, wherein R is selected from the group of 1), 2) or 3). Preferably,Y is O⁻Cat+, or a nucleoside. More preferably, Y is O⁻Cat+. Preferably,R1 is a methyl. Preferably, A is O or CH₂. More preferably, A is O.Preferably, n is 2. Preferably, X is a bromide. Preferably, B is O.Preferably, m is 1 or 2. More preferably, m is 1.

For example, synthetic γδT lymphocyte activators comprise the compoundsof formula (III) or (IV)

wherein X, R1, n, m and Y have the aforementioned meaning.

In one preferred embodiment, synthetic γδT lymphocyte activatorscomprise the compounds of formula (V):

in which X is an halogen (preferably selected from I, Br and Cl), R1 isa methyl or ethyl group, Cat+ represents one (or several, identical ordifferent) organic or mineral cation(s) (including the proton), and n isan integer from 2 to 20. Preferably, R1 is a methyl. Preferably, n is 2.Preferably, X is a bromide.

In a most preferred embodiment, synthetic γδT lymphocyte activatorscomprise the compound of formula (VI):

In an other most preferred embodiment, synthetic γδT lymphocyteactivators comprise the compound of formula (VII):

In one particular embodiment, synthetic γδT lymphocyte activatorscomprise the compounds of formula (VIII):

in which R1 is a methyl or ethyl group, Cat+ represents one (or several,identical or different) organic or mineral cation(s) (including theproton), B is O or NH, m is an integer from 1 to 3, and n is an integerfrom 2 to 20, A is O, NH, CHF, CF₂ or CH₂, and Y is O⁻Cat+, anucleoside, or a radical -A-R, wherein R is selected from the group of1), 2) or 3). Preferably, Y is O⁻Cat+, or a nucleoside. More preferably,Y is O⁻Cat+. Preferably, R1 is a methyl. Preferably, A is O or CH₂. Morepreferably, A is O. Preferably, n is 2. Preferably, B is O. Preferably,m is 1 or 2. More preferably, m is 1.

For example, synthetic γδT lymphocyte activators comprise the compoundsof formula (IX) or (X):

wherein R1, n, m and Y have the above mentioned meaning.

In one preferred embodiment, synthetic γδT lymphocyte activatorscomprise the compounds of formula (XI):

in which R1 is a methyl or ethyl group, Cat+ represents one (or several,identical or different) organic or mineral cation(s) (including theproton), and n is an integer from 2 to 20. Preferably, R1 is a methyl.Preferably, n is 2.

In a most preferred embodiment, synthetic γδT lymphocyte activatorscomprise the compound of formula (XI):

In one particular embodiment, synthetic γδT lymphocyte activatorscomprise the compounds of formula (XII):

in which R₃, R₄, and R₅, identical or different, are a hydrogen or(C₁-C₃)alkyl group, W is —CH— or —N—, R₆ is an (C₂-C₃)acyl, an aldehyde,an (C₁-C₃)alcohol, or an (C₂-C₃)ester, Cat+ represents one (or several,identical or different) organic or mineral cation(s) (including theproton), B is O or NH, m is an integer from 1 to 3, A is O, NH, CHF, CF₂or CH₂, and Y is O⁻Cat+, a nucleoside, or a radical -A-R, wherein R isselected from the group of 1), 2) or 3). Preferably, Y is O⁻Cat+, or anucleoside. More preferably, Y is O⁻Cat+. Preferably,. A is O or —CH₂.More preferably, A is O. More preferably, R₃ and R₅ are a methyl and R₄is a hydrogen. More preferably, R₆ is —CH₂—OH, —CHO, —CO—CH₃ or—CO—OCH₃. Preferably, B is O. Preferably, m is 1 or 2. More preferably,m is 1. Optionally, the double-bond between W and C is in conformationtrans (E) or cis (Z). More preferably, the double-bond between W and Cis in conformation trans (E).

For example, synthetic γδT lymphocyte activators comprise the compoundsof formula (XIII) or (XIV):

wherein R3, R4, R5, R6, W, m, and Y have the above mentionned meaning.Preferably, W is —CH—. Preferably, R3 and R4 are hydrogen. Preferably,R5 is a methyl. Preferably, R6 is —CH₂—OH.

In a most preferred embodiment, synthetic γδT lymphocyte activatorscomprise the compound of formula (XV):

In an other most preferred embodiment, synthetic γδT lymphocyteactivators comprise the compound of formula (XVI):

Specific examples of compounds include:

-   (E)1-pyrophosphonobuta-1,3-diene-   (E)1-pyrophosphonopenta-1,3-diene-   (E)1-pyrophosphono-4-methylpenta-1,3-diene-   (E,E)1-pyrophosphono4,8-dimethylnona-1,3,7-triene-   (E,E,E)1-pyrophosphono4,8,12-trimethyltrideca-1,3,7,11-tetraene-   (E,E)1-triphosphono4,8-dimethylnona-1,3,7-triene-   4-triphosphono-2-methylbutene-   α,β-di-[3-methylpent-3-enyl]-pyrophosphonate-   1-pyrophosphono-3-methylbut-2-ene-   α,γ-di-[3-methylbut-2enyl]-triphosphonate-   α,β-di-[3-methylbut-2-enyl]-pyrophosphonate-   allyl-pyrophosphonate-   allyl-triphosphonate-   α,γ-di-allyl-pyrophosphonate-   α,β-di-allyl-triphosphonate-   (E,E)4-[(5′-pyrophosphono-6′-methyl-penta-2′,4′-dienyloxymethyl)-phenyl]-phenyl-methanone-   (E,E)4-[(5′-triphosphono-6′-methyl-penta-2′,4′-dienyloxymethyl)-phenyl]-phenyl-methanone-   (E,E,E)[4-(9-pyrophosphono-2′,640    -dimethyl-nona-2′,6′,8′-trienyloxymethyl)-phenyl]-phenyl-methanone-   (E,E,E)[4-(9′-pyrophosphono-2′,6′,8′-trimethyl-nona-2′,6′,8′-trienyloxymethyl)-phenyl]-phenyl-methanone-   5-pyrophosphono-2-methypentene-   5-triphosphono-2-methypentene-   α,γ-di-[4-methylpent-4-enyl]-triphosphonate-   5-pyrophosphono-2-methypent-2-ene-   5-triphosphono-2-methypent-2-ene-   9-pyrophosphono-2,6-dimethynona-2,6diene-   9-triphosphono-2,6-dimethynona-2,6-diene-   α,γ-di-[4,8-dimethylnona-2,6-dienyl]-triphosphonate-   4-pyrophosphono-2-methybutene-   4methyl-2-oxa-pent-4-enyloxymethylpyrophosphate-   4-methyl-2-oxa-pent-4-enyloxymethyltriphosphate-   α,β-di-[4-methyl-2-oxa-pent-4enyloxymethyl]-pyrophosphate-   α,γ-di-[4-methyl-2-oxa-pent-4-enyloxymethyl]-triphosphate

Phosphohalohydrins (R=1^(st)))

-   3-(halomethyl)-3-butanol-1-yl-diphosphate-   3-(halomethyl)-3-pentanol-1-yl-diphsophate-   4-(halomethyl)4-pentanol-1-yl-diphosphate-   4-(halomethyl)-4hexanol-1-yl-diphosphate-   5-(halomethyl)-5-hexanol-1-yl-diphosphate-   5-(halomethyl)-5-heptanol-1-yl-diphosphate-   6-(halomethyl)6-heptanol-1-yl-diphosphate-   6-(halomethyl)-6-octanol-1-yl-diphosphate-   7-(halomethyl)-7-octanol-1-yl-diphosphate-   7-(halomethyl)-7-nonanol-1-yl-diphosphate-   8-(halomethyl)-8-nonanol-1-yl-diphosphate-   8-halomethyl)-8-decanol-1-yl-diphosphate-   9-(halomethyl)-9-decanol-1-yl-diphosphate-   9-(halomethyl)-9-undecanol-1-yl-diphosphate-   10-(halomethyl)-10-undecanol-1-yl-diphosphate-   10-(halomethyl)-10-dodecanol-1-yl-diphosphate-   11-(halomethyl)-11-dodecanol-1-yl-diphosphate-   11-(halomethyl)-11-tridecanol-1-yl-diphosphate-   12-(halomethyl)-12-tridecanol-1-yl-diphosphate-   12-(halomethyl)-12-tetradecanol-1-yl-diphosphate-   13-(halomethyl)-13-tetradecanol-1-yl-diphosphate-   13-(halomethyl)-13-pentadecanol-1-yl-diphosphate-   14-(halomethyl)-14-pentadecanol-1-yl-diphosphate-   14-(halomethyl)-14-hexadecanol-1-yl-diphosphate-   15-(halomethyl)-15-hexadecanol-1-yl-diphosphate-   15-(halomethyl)-15-heptadecanol-1-yl-diphosphate-   16-(halomethyl)-16-heptadecanol-1-yl-diphosphate-   16-(halomethyl)-16-octadecanol-1-yl-diphosphate-   17-(halomethyl)-17-octadecano1-yl-diphosphate-   17-(halomethyl)-17-nonadecanol-1-yl-diphosphate-   18-(halomethyl)-18-nonadecanol-1-yl-diphosphate-   18-(halomethyl)-18-eicosanol-1-yl-diphosphate-   19-(halomethyl)-19-eicosanol-1-yl-diphosphate-   19-(halomethyl)-19-heneicosanol-1-yl-diphosphate-   20-(halomethyl)-20-heneicosanol-1-yl-diphosphate-   20-(halomethyl)-20-docosanol-1-yl-diphosphate-   21-(halomethyl)-21-docosanol-1-yl-diphosphate-   21-(halomethyl)-21-tricosanol-1-yl-diphosphate

More particularly,

-   3-bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP)-   5-bromo-4-hydroxy4-methylpentyl pyrophosphonate (CBrHPP)-   3-iodomethyl)-3-butanol-1-yl-diphosphate (IHPP)-   3-chloromethyl)-3-butanol-1-yl-diphosphate (ClHPP)-   3-bromomethyl)-3-butanol-1-yl-triphosphate (BrHPPP)-   3-iodomethyl)-3-butanol-1-yl-triphosphate (IHPPP)-   α,γ-di-[3-(bromomethyl)-3-butanol-1-yl]-triphosphate (diBrHTP)-   α,γ-di-[3-(iodomethyl)-3-butanol-1-yl]-triphosphate (diIHTP)

Phosphoepoxydes (R=2^(nd)))

-   3,4-epoxy-3-methyl-1-butyl-diphosphate (Epox-PP)-   3,4,-epoxy-3-methyl-1-butyl-triphosphate (Epox-PPP)-   α,γ-di-3,4,-epoxy-3-methyl-1-butyl-triphosphate (di-Epox-TP)-   3,4-epoxy-3-ethyl-1-butyl-diphosphate-   4,5-epoxy4-methyl-1-pentyl-diphosphate-   4,5-epoxy-4-ethyl-1-pentyl-diphosphate-   5,6-epoxy-5-methyl-1-hexyl-diphosphate-   5,6-epoxy-5-ethyl-1-hexyl-diphosphate-   6,7-epoxy-6-methyl-1-heptyl-diphosphate-   6,7-epoxy-6-ethyl-1-heptyl-diphosphate-   7,8-epoxy-7-methyl-1-octyl-diphosphate-   7,8-epoxy-7-ethyl-1-octyl-diphosphate-   8,9-epoxy-8-methyl-1-nonyl-diphosphate-   8,9-epoxy-8-ethyl-1-nonyl-diphosphate-   9,10-epoxy-9-methyl-1-decyl-diphosphate-   9,10-epoxy-9-ethyl-1-decyl-diphosphate-   10,11-epoxy-10-methyl-1-undecyl-diphosphate-   10,11-epoxy-10-ethyl-1-undecyl-diphosphate-   11,12-epoxy-11-methyl-1-dodecyl-diphosphate-   11,12-epoxy-11-ethyl-1-dodecyl-diphosphate-   12,13-epoxy-12-methyl-1-tridecyl-diphosphate-   12,13-epoxy-12-ethyl-1-tridecyl-diphosphate-   13,14-epoxy-13-methyl-1-tetradecyl-diphosphate-   13,14-epoxy-13-ethyl-1-tetradecyl-diphosphate-   14,15-epoxy-14-methyl-1-pentadecyl-diphosphate-   14,15-epoxy-14-ethyl-1-pentadecyl-diphosphate-   15,16-epoxy-15-methyl-1-hexadecyl-diphosphate-   15,16-epoxy-15-ethyl-1-hexadecyl-diphosphate-   16,17-epoxy-16methyl-1-heptadecyl-diphosphate-   16,17-epoxy-16-ethyl-1-heptadecyl-diphosphate-   17,18-epoxy-17-methyl-1-octadecyl-diphosphate-   17,18-epoxy-17-ethyl-1-octadecyl-diphosphate-   18,19-epoxy-18-methyl-1-nonadecyl-diphosphate-   18,19-epoxy-18-ethyl-1-nonadecyl-diphosphate-   19,20-epoxy-19-methyl-1-eicosyl-diphosphate-   19,20-epoxy-19-ethyl-1-eicosyl-diphosphate-   20,21-epoxy-20-methyl-1-heneicosyl-diphosphate-   20,21-epoxy-20-ethyl-1-heneicosyl-phosphate-   21,22-epoxy-21-methyl-1-docosyl-diphosphate-   21,22-epoxy-21-ethyl-1-docosyl-diphosphate

More particularly,

-   3,4-epoxy-3-methyl-1-butyl-diphosphate (Epox-PP)-   3,4,-epoxy-3-methyl-1-butyl-triphosphate (Epox-PPP)-   α,γ-di-3,4,-epoxy-3-methyl-1-butyl-triphosphate (di-Epox-TP)-   uridine 5′-triphosphate -(3,4-époxy methyl butyl) (Epox-UTP)

Phosphoepoxydes (R=3^(rd))

-   (E)-4-hydroxy-3-methyl-2-butenyl pyrophosphate (HDMAPP)-   ()-5-hydroxy-4-methylpent-3-enyl pyrophosphonate (CHDMAPP)

These compounds may be produced according to various techniques knownper se in the art, some of which being disclosed in PCT Publicationsnos. WO 00/12516, WO 00/12519, WO 03/050128, and WO 03/009855, thedisclosures of which are incorporated herein by reference.

In a most preferred embodiment, the synthetic γδT lymphocyte activatingcompound is selected from the group consisting of HDMAPP, CHDMAPP,Epox-PP, BrHPP and CBrHPP, more preferably HDMAPP, CHDMAPP, BrHPP andCBrHPP, still more preferably HDMAPP.

Alternatively, although potentially less efficient, other activators foruse in the present invention are phosphoantigens disclosed in WO95/20673, isopentenyl pyrophosphate (IPP) (U.S. Pat. No. 5,639,653) and3-methylbut-3enyl pyrophosphonate (C-IPP). The disclosures of bothreferences are incorporated herein by reference.

Compounds comprising a nucleoside as Y group can be prepared, forexample, by the following reactions. Depending on the type andreactivity of the functional groups provided by Y, the professional isable to adapt the following examples, if necessary including the phasesof protection/non-protection of the sensitive functional groups or thosethat can interact with the coupling reaction.

where —O—V is a good group beginning with V chosen, for example, fromamong tosyle, mesyle, triflyle, brosyle or bromium, PP represents thepyrophosphate group, PPP represents the triphosphate group, R-A- has theabove mentionned meaning and Nucl is a nucleoside. Preferably, Nucl-O—Vis selected from the group consisting of: 5′-O-Tosyladenosine,5′-O-Tosyluridine, 5′-O-Tosylcytidine, 5′-O-Tosylthymidine or5′-O-Tosyl-2′-deoxyadenosine.

For example, for the compound with R of group 1), the reaction procedurecan be the following:

where —O—V is a good group beginning with V chosen, for example, fromamong tosyle, mesyle, triflyle, brosyle or bromium, PP represents thepyrophosphate group and Nucl is a nucleoside. Preferably, Nucl-O—V isselected from the group consisting of: 5′-O-Tosyladenosine,5′-O-Tosyluridine, 5′-O-Tosylcytidine, 5′-O-Tosylthymidine or5′-O-Tosyl-2′-deoxyadenosine as described in Davisson et al (1987), thedisclosure of which is incorporated herein by reference. Neutral pH is anucleophile substitution reaction that can be carried out in conditionssimilar to those described by Davisson et al, (1987); and Davisson etal. (1986), the disclosures of which are incorporated herein byreference.

This reaction can also be used to prepare compound comprising amonosaccharide as group Y. In this case, Nucl-O—V is replaced byMonoSac-O—V, wherein Monosac is monosaccharide. For example, it ispossible to use the MonoSac-O—Y group corresponding to compoundMethyl-6-O-tosyl-alpha-D-galactopyranoside as described in publicationNilsson and Mosbach, (1980), incorporated herein by reference, or thecommercially available mannose triflate compound.

This reaction can further be used to prepare compound comprising aoligosaccharide as group Y. In this case, Nucl-O—V is replaced byoligoSac-O—V, wherein oligoSac is an oligosaccharide. For example, it ispossible to use the oligoSac-O—Y group corresponding to compound6^(A)—O-p-Toluenesulfonyl-β-cyclodextrin as described in publication(Organic syntheses, Vol. 77, p 225-228, the disclosure of which isincorporated herein by reference).

This reaction can be used to prepare compound comprising apolysaccharide as group Y. In this case, Nucl-O—V is replaced bypolySac-O—V, wherein polySac is a polysaccharide. For example, it ispossible to use the polySac-O—Y group corresponding to tosylatedpolysaccharide as described in publication Nilsson et al., (1981); andNilsson and Mosbach, (1980), the disclosures of which are incorporatedherein by reference. This coupling technique based on the activation ofthe hydroxyl groups of a polysaccharide support by tosylation allows forcovalent coupling in an aqueous or an organic medium.

This reaction can also be used for preparing compound comprising analdehyde derivative as group Y by choosing, instead of Nucl, aderivative including a protected aldehyde function in the form of anacetal or any other group protecting this function.

Alternatively, compounds comprising a nucleoside as Y group can beprepared by the following reaction:

where PPP represents the triphosphate group, R-A has the abovementionned meaning, DMF is dimethylformamide, and Nucl is a nucleoside.This reaction can be carried out in conditions similar to thosedescribed by Knorre et al.(1976), or by Bloom et al., U.S. Pat. No.5,639,653 (1997), the disclosures of which are incorporated herein byreference, from alcohol and a nucleotide with formula Nucl-O-PPP.

For example, for the compound with R of group 1), the reaction procedurecan be the following:

where PPP represents the triphosphate group, DMF is dimethylformamide,and Nucl is a nucleoside.

This reaction can also be applied to the preparation of oligonucleotides5′-triphosphate ?-esters as indicated by the authors of publicationKnorre et al. (1976).

Compounds comprising a nucleic acid as Y group, more particularly aribonucleic acid, can be prepared in conditions similar to thosedescribed in publication F. Huang et al (1997). The authors describe auniversal method from catalytic RNA that is applicable to any moleculecomprising a free terminal phosphate group. Compounds structurallyrelated to the phosphohalohydrine group such as isopentenylpyrophosphate or thiamine pyrophosphate are used or mentioned by theseauthors (see p. 8968 of F. Huang et al (1997)). It should also be notedthat the experimental conditions for the coupling procedure (inparticular pH conditions) described in the section<<Reaction of Isolate6 pppRNA with phosphate containing Nucleophiles>> on page 8965 arecompatible with the presence of a halohydrine function.

Compounds comprising an amino acid, a peptide or a protein derivative asY group can be obtained using the well known reactivity of their primaryamine or thiol function on an epoxyde function (S_(N)2 reaction). Thistype of coupling classically involves an intermediate group still called“linker” bearing an epoxyde function. An example of a reaction procedureusing this type of coupling is provided below.

Reaction D where PP represents the pyrophosphate group, R-A has theabove mentionned meaning and R′—SH is an amino acid, a peptide or aprotein derivative. The first phase can be carried out in conditionssimilar to those described by Davisson et al. (1987) and Davisson et al,(1986), the disclosures of which are incorporated herein by reference,from the tetrabutylammonium salt of the initial compound andcommercially available compounds such as glycidyl tosylate orepichlorohydrine. This reaction can also be carried out withthriphosphate compounds. Alternatively, a primary amine R′—NH₂ can beused instead of R′—SH. Without the reaction with R′13 SH, the firstreaction can be used to prepare compound comprising an epoxydederivative.

Alternatively, compounds comprising an amino acid, a peptide or aprotein derivative as Y group can be prepared by the following reaction:

where PPP represents the triphosphate group, PP represents thepyrophosphate group, P represents the phosphate group, R-A has the abovementionned meaning and R′—NH is an amino acid, a peptide or a proteinderivative. The reaction can be carried out in conditions similar tothose described by Knorre et al. (1976), the disclosure of which isincorporated herein by reference, from compound (R-A-PPP) and an aminoacid, peptide or a protein with formula R—NH₂. This reaction involvesthe protection of the sensitive functions of compound R—NH₂ or can reactwith the carbodiimide (in particular, the carboxyl function).

Tri or tetra-n-butylammonium salts of phosphoric, pyrophosphoric,triphosphoric, tetra-phosphoric or polyphosphoric acid can be preparedfrom commercially available corresponding acids. Derivatives with arelated structure such as derivatives of methanetrisphosphonic aciddescribed in publication Liu et al (1999), the disclosure of which isincorporated herein by reference, can also be prepared according to thereaction procedure.

The above mentionned reactions can be extrapolated to a very largespectrum of molecules or biomolecules by using the reactivity of thehydroxyl, amine, phosphate or thiol functions. Thereby, inositolderivatives can be prepared according to reactions A or B by activationof the hydroxyl function. Derivatives of folic acid (vitamin B9) ortetrahydrofolic acid can be prepared according to reactions D or E bycalling on the reactivity of the primary amine function.

Of course, other types of coupling can be considered and theprofessional can have access to a large choice of reactions.

Thereby, coupling by phosphorylation of carboxylic acid or phenol groupscan be used for the formation of fatty acid, lipid or certain flavonoidderivatives.

The Cytokine

As indicated above, the method is based on the use of particularcombinations of active agents, according to particular schedules. Theinvention more preferably uses a cytokine in combination with asynthetic activator, the cytokine being an interleukin-2 polypeptide.

The interleukin-2 polypeptide may be of human of animal origin,preferably of human origin. It may comprise the sequence of a wild-typehuman (or anal) IL-2 protein, or any biologically active fragment,variant or analogue thereof, i.e., any fragment, variant or analoguecapable of binding to an IL-2 receptor and of inducing activation of γδTcells in the method of this invention.

The sequence of reference, wild-type human interleukin-2 proteins isavailable in the art, such as in Genbank under accession numbersNP000577; AAK26665; PO1585; XP035511, for instance, the disclosures ofwhich are incorporated herein by reference.

The term “variant” designates, in particular, any natural variants, suchas those resulting from polymorphism(s), splicing(s), mutation(s), etc.Such naturally-occurring variants may thus comprise one or severalmutation, deletion, substitution and/or addition of one or more aminoacid residues, as compared to a reference IL-2 protein sequence.

The term “variant” also includes IL-2 polypeptides originating fromvarious mammalian species, such as for instance rodent, bovine, porcine,equine, etc. More preferably, the IL-2 polypeptide is of human origin,i.e., comprises the sequence of a human IL-2 protein or a variant,fragment or analogue thereof.

The term “variant” also includes synthetic IL-2 variants, such as anysynthetic polypeptide comprising one or several mutation, deletion,substitution and/or addition of one or more amino acid residues, ascompared to a reference IL-2 protein sequence, and capable of binding toan IL-2 receptor and of inducing activation of γδT cells in the methodof this invention. Preferred synthetic IL-2 variants have at least 75%identity in amino acid sequence with the primary sequence of an IL-2reference protein, more preferably at least 80%, even more preferably atleast 85 or 90%. The identity between sequences may be determinedaccording to various known methods such as, typically, using the CLUSTALmethod.

Variants also include IL-2 polypeptides encoded by a nucleic acidsequence that hybridize, under 5 conventional, moderate stringency, withthe nucleic acid sequence encoding a reference IL-2 protein, or afragment thereof. Hybridization conditions are, for instance incubationat 40-42° C. for 12 hours in 50% formamide, 5×SSPE, 5× Denhardt'ssolution, 0.1% SDS.

The IL-2 polypeptide may also be any fragment of a reference IL-2protein which retain the ability to bind to an IL-2 receptor and toinduce activation of γδT cells in the method of this invention. Suchfragments contain, at least, one functional domain of IL-2, such as thereceptor binding site. Fragments contain preferably at least 40%, 50%or, preferably, at least 60% of the complete reference sequence.

Analogues designate polypeptides using the same receptor asInterleukin-2 and thus mediating similar activation signal in a γδlymphocyte.

The interleukin-2 polypeptide may further comprise heterologous residuesadded to the natural sequence, such as additional amino acids, sugar,lipids, etc. This may also be chemical, enzymatic or marker (e.g.,radioactive) groups.-The added residues or moiety may represent astabilizing agent, a transfection-facilitating agent, etc.

The IL-2 polypeptides may be in soluble, purified form, or conjugated orcomplexed with an other molecule, such as a biologically active peptide,protein, lipid, etc. The IL-2 polypeptide may be produced according totechniques known in the art, such as by chemical synthesis, enzymaticsynthesis, genetic (e.g., recombinant DNA) synthesis, or a combinationthereof. An IL-2 polypeptide of pharmaceutical grade may also beobtained from commercial sources.

The interleukin-2 polypeptide is preferably administered at low doses,i.e. at doses that are sufficient to target in vivo cells that expressthe high affinity receptor for IL2, defined as the tri-molecular complexCD25/CD122/CD130. Practically, in human, such doses have beenexperimentally defined in clinical trials as being comprised between 0.2and 2 million units per square meters, when injected subcutaneously (seefor example buzio et al 2001).

The IL-2 polypeptide is preferably administered by injection of between0.1 and 3 Million Units per day, over a period of 1 to 10 days.Preferably, daily doses of between 0.2 and 2 MU per day, even morepreferably between 0.2 and 1.5 MU, further preferably between 0.2 and 1MU, are being administered The daily dose may be administered as asingle injection or in several times, typically in two equal injections.The IL-2 treatment is preferably maintained over between 1 and 9 days,even more preferably during 3 to 7 days. Optimum effect seems to beachieved after 5 days treatment.

PREFERRED EMBODIMENTS OF THE INVENTION

In preferred embodiments, compounds BrHPP, CBrHPP, HDMAPP, CHDMAPP andepoxPP are used according to the methods of the invention.

BrHPP and EpoxPP

The synthesis of BrHPP is described in Example 1 and in Espinosa (2001),the disclosure of which is incorporated herein by reference. Thesynthesis of EpoxPP is described in European Patent No. 1109818B1, thedisclosure of which is incorporated herein by reference.

(1) The present invention relates especially to the treatment of adisease, especially a tumor, especially a solid tumor, more especiallyone of the preferred diseases as defined above or below, characterizedin that a compound of formula II, II or VII, especially BRHPP or EpoxPP,is administered more than once, with a two-weekly up to eight-weekly,preferably between three-weekly or four-weekly interval to a human in adose that is calculated according to the formula (A)single dose (mg/kg)=(0.1 to y)*N   (A)where N (a whole or fractional number) is the number of weeks betweentreatments (about two to about eight weeks), that is N is about 2 toabout 8, preferably between about 3 to 4; more preferably, the treatmentdose is calculated according to the formula B,single dose (mg/kg)=(5 to 100)* N;   (B)even more preferably according to the formula C,single dose (mg/kg)=(10 to 100)*N;   (C)or still more preferably according to the formula D,single dose (mg/m2)=(5 to 60)*N   (D)where, in each of formulae A to D, N is about 2 to about 8 or preferablyabout 3 to 4 (corresponding to intervals of about 2 to about 8 weeks andabout 3 to about 4 weeks between treatments);the compound of formula II or A, especially BRHPP, administrationpreferably taking place:

(a) about three-weekly to about four weekly, preferably three-weekly orfour-weekly, in a human in a dose that lies between about 0.1 mg/kg andabout 1.2 g/kg, preferably between about 10 mg/kg and about 1.2 g/kg,more preferably between about 5 mg/kg and about 100 mg/kg, even morepreferably between about 5 mg/kg and 60 mg/kg, or preferably about 20mg/kg, or

(b) about four-weekly to about eight weekly, preferably aboutfive-weekly, six-weekly, seven-weekly or eight-weekly, in a human in adose that lies dose is between about between about 0.1 mg/kg and about1.2 g/kg, preferably between about 10 mg/kg and about 1.2 g/kg, morepreferably between about 5 mg/kg and about 100 mg/kg, even morepreferably between about 5 mg/kg and 60 mg/kg, or preferably about 20mg/kg;

the administration preferably taking place by i.v. infusion during 2 to120 min, more preferably during about 5 to about 30 min, most preferablyduring about 10 to about 30 min, e.g. during about 30 min.

(2) The present invention preferably relates also to the treatment of atumor disease, most preferably a tumor disease having metastases, saidtumor being selected from a gastrointestinal, e.g. colorectal; lungtumor, especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor; a renal; a genitouriay, e.g. prostatic; a pancreatic;and a brain tumor (and/or any metastasis thereof), most preferably agastrointestinal tumor, especially a colorectal cancer, more especiallya gastrointestinal cancer, especially a colorectal cancer; or a tumor ofthe genitourinary tract, especially a prostate cancer; where a compoundof formula II, III or VIII, especially BRHPP or EpoxPP, is administeredto a warm-blooded animal, especially a human.

(3) The present invention also preferably relates to an in vivo regimenfor stimulating a γδ T cell in an individual, preferably a regimen fortreatment of a tumor disease, preferably a solid tumor, or an autoimmunedisorder or an infectious disease; wherein a composition is administeredto an individual such that a compound of formula II, III or VIII,especially BRHPP or EpoxPP is administered once in a dose that is

(a) between about the EC50 value and the EC100 value, more preferably atleast 110%, 120%, 150% or 175% of the EC50, to a human

(b) between about 0.1 mg/kg and about 100 mg/kg, to a human

and, if required, one or more (preferably at least two, at least three,at least four, at least five, at least six, at least eight or at leastten) further doses each within the dose range mentioned above for thefirst dose are administered in farther treatment cycles, preferably eachdose after a period of time that allows for sufficient recovery of theγδ T cell population to basal levels in the treated individual from eachpreceding dose administration, especially more than one week, more thantwo weeks after the preceding treatment, more especially two to eightweeks, most especially three to four weeks after the precedingtreatment, especially three weeks after that treatment.

More preferably, under (1) to (3) a compound of formula I, III or VIII,especially BRHPP or EpoxPP is administered three-weekly to a human in adose that lies between about 0.1 mg/kg and about 1.2 g/kg, preferablybetween about 10 mg/kg and about 1.2 g/kg, more preferably between about5 mg/kg and about 100 mg/kg, even more preferably between about 5 mg/kgand 60 mg/kg, or preferably about 20 mg/kg; or a compound of formula II,III or VIII, especially BRHPP or EpoxPP is administered four-weekly(every 4 weeks) in a dose that is between about 0.1 mg/kg and about 1.2g/kg, preferably between about 10 mg/kg and about 1.2 g/kg, morepreferably between about 5 mg/kg and about 100 mg/kg, even morepreferably between about 5 mg/kg and 60 mg/kg, or preferably about 20mg/kg;. This dose is preferably administered to the human by intravenous(i.v.) administration during 2 to 120 min, more preferably during about5 to about 30 min, most preferably during about 10 to about 30 min, e.g.during about 30 min.

More preferably, said treatment is repeated until disease progression,unacceptable toxicity, 1 or preferably 2 cycles beyond determination ofa complete response, or patient withdrawal of consent for any reason isencountered.

(4) The present invention preferably also relates to an in vivo regimenfor the treatment of a tumor disease, especially (i) of a solid tumorselected from a gastrointestinal e.g. colorectal; lung tumor, especiallya non-small cell lung carcinoma; a breast tumor; an epidermoid tumor, arenal; a genitourinary, e.g. prostatic; a pancreatic; and a brain tumor(and/or any metastasis thereof), most preferably a gastrointestinaltumor, especially a colorectal cancer, more especially agastrointestinal cancer, especially a colorectal cancer, or a tumor ofthe genitourinary tract, especially a prostate cancer, especially wheresuch tumor is metastatic, wherein a compound of formula II, III or VII,especially BRHPP or EpoxPP, is administered between once-weekly andeight-weekly to a warm-blooded animal in a dose that is below 80%, morepreferably below 50% of the maximal tolerable dose (AM) or highest dosetested in non-human animals.

Preferably, in the case of weekly treatment of a human with saidcompound of formula II, III or VIII, especially BRHPP or EpoxPP, thedose is in the range of about 1 to about 60%, preferably about 10 toabout 60%, e.g. about 5 to about 35% of the MTD, for example in therange of about 30 to about 35% of the MTD. Preferably, for BRHPP thedose is in the range of about 5 to about 60%, preferably about 10 toabout 60%, especially in the range of about 10 to about 45%, mostespecially in the range of about 30 to about 45% of the MTD.

(5) The present invention preferably also relates to an in vivo regimenfor the treatment of a disease, especially a solid tumor diseaseselected from a gastrointestinal, e.g. colorectal; lung tumor,especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor; a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer; or a tumor of the genitourinary tract, especially a prostatecancer; especially where such tumor is metastatic, wherein a compound offormula II, III or VIII, especially BRHPP or EpoxPP, is administeredbetween once-weekly and eight-weekly to a warm-blooded animal in a dosethat is between the Efficient Concentration value giving half themaximum effect (EC50) and the Efficient Concentration value giving themaximal effect (EC100), or that is between the EC50 and 200% of theEC50, or preferably at least 110%, 120%, 130%, 150%, 160%, 175% or 200%of the EC50 value.

CBrHPP

The synthesis of CBrHPP can be carried according to any suitable method.In a preferred example, 3-methylbut-3-enyl pyrophosphonate (C-IPP) isprepared according to the methods of PCT patent publication no. WO03/050128, Brondino et al, (1996), or Valentijn et al. (1991), and isconverted to CBrHPP according to the methods of Espinosa et al (2001a).Each of the cited references is incorporated herein by reference.

(1) The present invention relates especially to the treatment of adisease, especially a tumor, especially a solid tumor, more especiallyone of the preferred diseases as defined above or below, characterizedin that a CBrHPP compound is administered more than once, with atwo-weekly up to eight-weekly, preferably between three-weekly andfour-weekly interval to a human in a dose that is calculated accordingto the formula (A)single dose (mg/kg)=(0.1 to y)*N   (A)where N (a whole or fractional number) is the number of weeks betweentreatments (about two to about eight weeks), that is N is about 2 toabout 8, preferably between about 3 to 4; more preferably, the treatmentdose is calculated according to the formula B,single dose (mg/kg)=(5 to 100)*N;   (B)even more preferably according to the formula C,single dose (mg/kg)=(10 to 100)*N;   (C)or still more preferably according to the formula D,single dose (mg/m2)=(5 to 60)*N   (D)where, in each of formulae A to D, N is about 2 to about 8 or preferablyabout 3 to 4 (corresponding to intervals of about 2 to about 8 weeks andabout 3 to about 4 weeks between treatments);the CBrHPP administration preferably taking place:

(a) about three-weekly to about four weekly, preferably three-weekly orfour-weekly, in a human in a dose that lies between about 0.1 mg/kg andabout 1.2 g/kg, preferably between about 10 mg/kg and about 1.2 g/kg,more preferably between about 5 mg/kg and about 100 mg/kg, even morepreferably between about 5 mg/kg and 60 mg/kg, or preferably about 20mg/kg; or

(b) about four-weekly to about eight weekly, preferably aboutfive-weekly, six-weekly, seven-weekly or eight-weekly, in a human in adose that lies dose is between about 0.1 mg/kg and about 1.2 g/kg,preferably between about 10 mg/kg and about 1.2 g/kg, more preferablybetween about 5 mg/kg and about 100 mg/kg, even more preferably betweenabout 5 mg/kg and 60 mg/kg, or preferably about 20 mg/kg;

the admiration preferably taking place by i.v. infusion during 2 to 120min, more preferably during about 5 to about 30 min, most preferablyduring about 10 to about 30 min, e.g. during about 30 min.

(2) The present invention preferably relates also to the treatment of atumor disease, most preferably a tumor disease having metastases, saidtumor being selected from a gastrointestinal e.g. colorectal; lungtumor, especially a non-small cell lung carcinoma; a breast tumor, anepidermoid tumor; a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer, or a tumor of the genitourinary tract, especially a prostatecancer; wherein CBrHPP is administered to a warm-blooded animal,especially a human.

(3) The present invention also preferably relates to an in vivo regimenfor stimulating a γδ T cell in an individual, preferably a regimen fortreatment of a tumor disease, preferably a solid tumor, or an autoimmunedisorder or an infectious disease; wherein CBrHPP is administered oncein a dose that is

(a) between about the EC50 value and the EC100 value, more preferably atleast 110%, 120%, 150% or 175% of the EC50, to a human

(b) between about 0.1 mg/kg and about 100 mg/kg, to a human

and, if required, one or more (preferably at least two, at least three,at least four, at least five, at least six, at least eight or at leastten) further doses each within the dose range mentioned above for thefirst dose are administered in further treatment cycles, preferably eachdose after a period of time that allows for sufficient recovery of theγδ T cell population to basal levels in the treated individual from eachpreceding dose administration, especially more than one week, more thantwo weeks after the preceding treatment, more especially two to eightweeks, most especially three to four weeks after the precedingtreatment, especially three weeks after that treatment.

More preferably, under (1) to (3) CBrHPP is administered three-weekly toa human in a dose that lies between about 0.1 mg/kg and about 1.2 g/kg,preferably between about 10 mg/kg and about 1.2 g/kg, more preferablybetween about 5 mg/kg and about 100 mg/kg, even more preferably betweenabout 5 mg/kg and 60 mg/kg, or preferably about 20 mg/kg; or CBrHPP isadministered four-weekly (every 4 weeks) in a dose that is between about0.1 mg/kg and about 1.2 g/kg, preferably between about 10 mg/kg andabout 1.2 g/kg, more preferably between about 5 mg/kg and about 100mg/kg, even more preferably between about 5 mg/kg and 60 mg/kg, orpreferably about 20 mg/kg. This dose is preferably administered to thehuman by intravenous (i.v.) administration during 2 to 120 min, morepreferably during about 5 to about 30 min, most preferably during about10 to about 30 min, e.g. during about 30 min.

More preferably, said treatment is repeated until disease progression,unacceptable toxicity, 1 or preferably 2 cycles beyond determination ofa complete response, or patient withdrawal of consent for any reason isencountered.

(4) The present invention preferably also relates to an in vivo regimenfor the treatment of a tumor disease, especially (i) of a solid tumorselected from a gastrointestinal, e.g. colorectal; lung tumor,especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor, a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer, or a tumor of the genitourinary tract, especially a prostatecancer, especially where such tumor is metastatic, wherein CBrHPP, isadministered between once-weekly and eight-weekly to a warm-bloodedanimal in a dose that is below 80%, more preferably below 50% of themaximal tolerable dose (MTD).

Preferably, in the case of weekly treatment of a human with said CBrHPPcompound, the dose is in the range of about 1 to about 60%, preferablyabout 10 to about 60%, e.g. about 5 to about 35% of the MTD, for examplein the range of about 30 to about 35% of the MTD. Preferably, for CBrHPPthe dose is in the range of about 5 to about 60%, preferably about 10 toabout 60%, especially in the range of about 10 to about 45%, mostespecially in the range of about 30 to about 45% of the MTD. In aspecial case, the dose can be between about 2 and about 18 mg/m2 forCBrHPP.

(5) The present invention preferably also relates to an in vivo regimenfor the treatment of a disease, especially a solid tumor diseaseselected from a gastrointestinal, e.g. colorectal; lung tumor,especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor, a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer; or a tumor of the genitourinary tract, especially a prostatecancer; especially where such tumor is metastatic, wherein CBrHPP isadministered between once-weekly and eight-weekly to a warm-bloodedanimal in a dose that is between the Efficient Concentration valuegiving half the maximum effect (EC50) and the Efficient Concentrationvalue giving the maximal effect (EC100), or that is between 110% and200% of the EC50, or preferably at least 110%, 120%, 130%, 150%, 160%,175% or 200% of the EC50 value.

HDMAPP

Since the isolation of HDMAPP from E. coli cells deficient in the lytBcomponent of the non-mevalonate (MEP) pathway, described in Hintz et al(2001), the chemical synthesis of HDMAPP has been achieved by a numberof laboratories. The synthetic HDMAPP and the natural compound isolatedfrom E. coli lytB mutants displayed identical activities in stimulatingVγ9/Vδ2 T cells. The reactivity of human peripheral blood mononuclearceUs towards HDMAPP was restricted Vγ9/Vδ2 T ceUs, leading toup-regulation of activation markers on the cell surface, secretion ofpro-inflammatory cytokines, and expansion of the Vγ9/Vδ2 subpopulationin the presence of co-stimulation provided by IL-2. HDMAPP was reportedto have an EC₅₀ value of approx. 0.1 nM (compared to IPP with an EC₅₀ ofapprox. 1 μM), leading to the assumption that HDMAPP exclusivelyaccounted for the known Vγ9/Vδ2 T cell reactivity towards pathogenicbacteria such as Brucella, Campylobacter, Ehrlichia, E. coli,Francisella, Listeria, Mycobacterium, Pseudomonas, Salmonella, andYersinia, as well as to the protozoan parasites Plasmodium andToxoplasma.

In vitro in vivo pharmacodynamics of Vγ9/Vδ2⁺ T cell stimulation aselucidated by the inventors have now shown that HDMAPP is surprisinglyeffective in regulating γδ cell activity, and may be administered tomammals in a low dose administration regimens.

Preferred methods for the synthesis of HDMAPP are described in Example 2and in Wolff et al, Tetrahedron Letters (2002) 43:2555 and Hecht et al,Tetrahedron Letters (2002) 43: 8929, the disclosures of which areincorporated herein by reference for their teaching of methods ofpreparing HDMAPP compounds.

(1) The present invention relates especially to the treatment of adisease, especially a tumor, especially a solid tumor, more especiallyone of the preferred diseases as defined above or below, characterizedin that a compound of formula XII, especially HDMAPP, is administeredmore than once, with a two-weekly up to eight-weekly, preferably betweenthree-weekly and four-weekly interval to a human in a dose that iscalculated according to the formula (A)single dose (mg/kg)=(0.1 to y)*N   (A)where N (a whole or fractional number) is the number of weeks betweentreatments (about two to about eight weeks), that is N is about 2 toabout 8, preferably between about 3 to 4; more preferably, the treatmentdose is calculated according to the formula B,single dose (mg/kg)=(0.001 to 100)*N;   (B)even more preferably according to the formula C,single dose (mg/kg)=(0.01 to 5)*N;   (C)or still more preferably according to the formula D,single dose (mg/m2)=(0.02 to 2.5)*N   (D)where, in each of formulae A to D, N is about 2 to about 8 or preferablyabout 3 to 4 (corresponding to intervals of about 2 to about 8 weeks andabout 3 to about 4 weeks between treatments);the a compound of formula XII, especially HDMAPP, administrationpreferably taking place:

(a) about three-weekly to about four weekly, preferably three-weekly orfour-weekly, in a human in a dose that lies between about 1 μg/kg andabout 100 mg/kg, preferably between about 10 μg/kg and about 20 mg/kg,more preferably between about 20 μg/kg and about 5 mg/kg, even morepreferably between about 20 μg/kg and 2.5 mg/kg, or preferably about 0.5mg/kg, or preferably about 0.5 mg/kg; or

(b) about four-weekly to about eight weekly, preferably aboutfive-weekly, six-weekly, seven-weekly or eight-weekly, in a human in adose that lies dose is between about 1 μg/kg and about 100 mg/kg,preferably between about 10 μg/kg and about 20 mg/kg, more preferablybetween about 20 μg/kg and about 5 mg/kg, even more preferably betweenabout 20 μg/kg and 2.5 mg/kg, or preferably about 0.5 mg/kg, orpreferably about 0.5 mg/kg; the administration preferably taking placeby i.v. infusion during 2 to 120 min, more preferably during about 5 toabout 30 min, most preferably during about 10 to about 30 min, e.g.during about 30 min.

(2) The present invention preferably relates also to the treatment of atumor disease, most preferably a tumor disease having metastases, saidtumor being selected from a gastrointestinal, e.g. colorectal; lungtumor, especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor; a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer; or a tumor of the genitourinary tract, especially a prostatecancer; a said compound of formula XII, especially HDMAPP, isadministered to a warm-blooded animal, especially a human.

(3) The present invention also preferably relates to an in vivo regimenfor stimulating a γδ T cell in an individual, preferably a regimen fortreatment of a tumor disease, preferably a solid tumor, or an autoimmunedisorder or an infectious disease; wherein a compound of formula XII,especially HDMAPP is administered once in a dose that is

(a) between about the EC50 and the EC100, more preferably at least 110%,120%, 150% or 175% of the EC50, to a human

(b) between about 10 μg/kg and about 20 mg/kg, to a human

and, if required, one or more (preferably at least two, at least three,at least four, at least five, at least six, at least eight or at leastten) further doses each within the dose range mentioned above for thefirst dose are administered in further treatment cycles, preferably eachdose after a period of time that allows for sufficient recovery of theγδ T cell population to basal levels in the treated individual from eachpreceding dose administration, especially more than one week, more thantwo weeks after the preceding treatment, more especially two to eightweeks, most especially three to four weeks after the precedingtreatment, especially three weeks after that treatment.

More preferably, under (1) to (3) a compound of formula XII, especiallyHDMAPP is administered three-weekly to a human in a dose that liesbetween about 1 μg/kg and about 100 mg/kg, preferably between about 10μg/kg and about 20 mg/kg, more preferably between about 20 μg/kg andabout 5 mg/kg, even more preferably between about 20 μg/kg and 2.5mg/kg, or preferably about 0.5 mg/kg, or preferably about 0.5 mg/kg; ora compound of formula XII, especially HDMAPP is administered four-weekly(every 4 weeks) in a dose that is between about 1 μg/kg and about 100mg/kg, preferably between about 10 μg/kg and about 20 mg/kg, morepreferably between about 20 μg/kg and about 5 mg/kg, even morepreferably between about 20 μg/kg and 2.5 mg/kg, or preferably about 0.5mg/kg, or preferably about 0.5 mg/kg. This dose is preferablyadministered to the human by intravenous (i.v.) administration during 2to 120 min, more preferably during about 5 to about 30 min, mostpreferably during about 10 to about 30 min, e.g. during about 30 min.

More preferably, said treatment is repeated until disease progression,unacceptable toxicity, 1 or preferably 2 cycles beyond determination ofa complete response, or patient withdrawal of consent for any reason isencountered.

(4) The present invention preferably also relates to an in vivo regimenfor the treatment of a tumor disease, especially (i) of a solid tumorselected from a gastrointestinal, e.g. colorectal; lung tumor,especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor; a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metasasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer, or a tumor of the genitourinary tract, especially a prostatecancer, especially where such tumor is metastatic, wherein a compound offormula XII, especially HDMAPP, is administered between once-weekly andeight-weekly to a warm-blooded animal in a dose that is below 80%, morepreferably below 50% of the maximal tolerable dose (MTD).

Preferably, in the case of weekly treatment of a human with said acompound of formula XII, especially HDMAPP, the dose is in the range ofabout 1 to about 60%, preferably about 10 to about 60%, e.g. about 5 toabout 35% of the MTD, for example in the range of about 30 to about 35%of the MTD. Preferably, for HDMAPP the dose is in the range of about 5to about 60%, preferably about 10 to about 60%, especially in the rangeof about 10 to about 45%, most especially in the range of about 30 toabout 45% of the MTD. In a special case, the dose can be between about 2and about 18 mgtm2 for HDMAPP.

(5) The present invention preferably also relates to an in vivo regimenfor the treatment of a disease, especially a solid tumor diseaseselected from a gastrointestinal, e.g. colorectal; lung tumor,especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor; a renal; a genitouriay, e.g. prostatic; a pancreatic;and a brain tumor (and/or any metastasis thereof), most preferably agastrointestinal tumor, especially a colorectal cancer, more especiallya gastrointestinal cancer, especially a colorectal cancer; or a tumor ofthe genitourinary tract, especially a prostate cancer; especially wheresuch tumor is metastatic, wherein a compound of formula XII, especiallyHDMAPP, is administered between once-weekly and eight-weekly to awarm-blooded animal in a dose that is between the EfficientConcentration value giving half the maximum effect (EC50) and theEfficient Concentration value giving the maximal effect (EC100), or thatis between 110% and 200% of the EC50, or preferably at least 110%, 120%,130%, 150%, 160%, 175% or 200% of the EC50 value.

CHDMAPP

The synthesis of CHDMAPP can be carried according to any suitablemethod. Examples include the methods of Nakamura et al (1973), Zoreticand Zhang (1996) or Umbreit and Sharpless (1977), the disclosures ofwhich are incorporated herein by reference, to produce aE-hydroxydimethylallyl type synthon prior to phosphorylation ouphosphonation. Phosphorylation or phosphonation can then be carried outaccording to methods described in PCT patent publication no. WO03/050128, Brondino et al, (1996), or

Valentijn et al.(1991), the disclosures of which are incorporated hereinby reference.

(1) The present invention relates especially to the treatment of adisease, especially a tumor, especially a solid tumor, more especiallyone of the preferred diseases as defined above or below, characterizedin that a CHDMAPP compound is administered more than once, with atwo-weekly up to eight-weekly, preferably between three-weekly andfour-weekly interval to a human in a dose that is calculated accordingto the formula (A)single dose (mg/kg)=(0.1 to y)*N   (A)where N (a whole or fractional number) is the number of weeks betweentreatments (about two to about eight weeks), that is N is about 2 toabout 8, preferably between about 3 to 4; more preferably, the treatmentdose is calculated according to the formula B,single dose (mg/kg)=(5 to 100)*N;   (B)even more preferably according to the formula C,single dose (mg/kg)=(10to 100)*N;   (C)or still more preferably according to the formula D,single dose (mg/m2)=(5 to 60)*N   (D)where, in each of formulae A to D, N is about 2 to about 8 or preferablyabout 3 to 4 (corresponding to intervals of about 2 to about 8 weeks andabout 3 to about 4 weeks between treatments);the CHDMAPP administration preferably taking place:

(a) about three-weekly to about four weekly, preferably three-weekly orfour-weekly, in a human in a dose that lies between about 1 μg/kg andabout 100 mg/kg, preferably between about 10 μg/kg and about 20 mg/kg,more preferably between about 20 μg/kg and about 5 mg/kg, even morepreferably between about 20 μg/kg and 2.5 mg/kg, or preferably about 0.5mg/kg, or preferably about 0.5 mg/kg; or

(b) about four-weekly to about eight weekly, preferably aboutfive-weekly, six-weekly, seven-weekly or eight-weekly, in a human in adose that lies dose is between about 1 μg/kg and about 100 mg/kg,preferably between about 10 μg/kg and about 20 mg/kg, more preferablybetween about 20 μg/kg and about 5 mg/kg, even more preferably betweenabout 20 μg/kg and 2.5 mg/kg, or preferably about 0.5 mg/kg, orpreferably about 0.5 mg/kg,

the administration preferably taking place by i.v infusion during 2 to120 min, more preferably during about 5 to about 30 min, most preferablyduring about 10 to about 30 min, e.g. during about 30 min.

(2) The present invention preferably relates also to the treatment of atumor disease, most preferably a tumor disease having metastases, saidtumor being selected from a gastrointestinal, e.g. colorectal; lungtumor, especially a non-small cell lung carcinoma; a breast tumor, anepidermoid tumor, a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer; or a tumor of the genitourinary tract, especially a prostatecancer, where CHDMAPP is administered to a warm-blooded animal,especially a human.

(3) The present invention also preferably relates to an in vivo regimenfor stimulating a γδ T cell in an individual, preferably a regimen fortreatment of a tumor disease, preferably a solid tumor, or an autoimmunedisorder or an infectious disease; wherein CHDMAPP is administered oncein a dose that is

(a) between about the EC50 and the EC100, more preferably at least 110%,120%, 150% or 175% of the ECS0, to a human

(b) between about 10 μg/kg and about 20 mg/kg, to a human

and, if required, one or more (preferably at least two, at least three,at least four, at least five, at least six, at least eight or at leastten) further doses each within the dose range mentioned above for thefirst dose are administered in further treatment cycles, preferably eachdose after a period of time that allows for sufficient recovery of theγδ T cell population to basal levels in the treated individual from eachpreceding dose administration, especially more than one week, more thantwo weeks after the preceding treatment, more especially two to eightweeks, most especially three to four weeks after the precedingtreatment, especially three weeks after that treatment.

More preferably, under (1) to (3) CHDMAPP is administered three-weeklyto a human in a dose that lies between about 1 μg/kg and about 100mg/kg, preferably between about 10 μg/kg and about 20 mg/kg, morepreferably between about 20 μg/kg and about 5 mg/kg, even morepreferably between about 20 μg/kg and 2.5 mg/kg, or preferably about 0.5mg/kg, or preferably about 0.5 mg/kg, or CHDMAPP is administeredfour-weekly (every 4 weeks) in a dose that is between about 1 μg/kg andabout 100 mg/kg, preferably between about 10 μg/kg and about 20 mg/kg,more preferably between about 20 μg/kg and about 5 mg/kg, even morepreferably between about 20 μg/kg and 2.5 mg/kg, or preferably about 0.5mg/kg, or preferably about 0.5 mg/kg. This dose is preferablyadministered to the human by intravenous (i.v.) administration during2.to 120 min, more preferably during about 5 to about 30 min, mostpreferably during about 10 to about 30 min. e.g. during about 30 min.

More preferably, said treatment is repeated until disease progression,unacceptable toxicity, 1 or preferably 2 cycles beyond determination ofa complete response, or patient withdrawal of consent for any reason isencountered.

(4) The present invention preferably also relates to an in vivo regimenfor the treatment of a tumor disease, especially (i) of a solid tumorselected from a gastrointestinal e.g. colorectal; lung tumor, especiallya non-small cell lung carcinoma; a breast tumor, an epidermoid tumor, arenal; a genitourinary, e.g. prostatic; a pancreatic; and a brain tumor(and/or any metastasis thereof), most preferably a gastrointestinaltumor, especially a colorectal cancer, more especially agastrointestinal cancer, especially a colorectal cancer; or a tumor ofthe genitourinary tract, especially a prostate cancer; especially wheresuch tumor is metastatic, wherein CHDMAPP is administered betweenonce-weekly and eight-weekly to a warm-blooded animal in a dose that isbelow 80%, more preferably below 50% of the maximal tolerable dose(MTD).

Preferably, in the case of weekly treatment of a human with said CHDMAPPthe dose is in the range of about 1 to about 60%, preferably about 10 toabout 60%, e.g. about 5 to about 35% of the MTD, for example in therange of about 30 to about 35% of the MTD. Preferably, for CHDMAPP thedose is in the range of about 5 to about 60%, preferably about 10 toabout 60%, especially in the range of about 10 to about 45%, mostespecially in the range of about 30 to about 45% of the MTD. In aspecial case, the dose can be between about 2 and about 18 mgtm2 forCHDMAPP.

(5) The present invention preferably also relates to an in vivo regimenfor the treatment of a disease, especially a solid tumor diseaseselected from a gastrointestinal, e.g. colorectal; lung tumor,especially a non-small cell lung carcinoma; a breast tumor; anepidermoid tumor; a renal; a genitourinary, e.g. prostatic; apancreatic; and a brain tumor (and/or any metastasis thereof), mostpreferably a gastrointestinal tumor, especially a colorectal cancer,more especially a gastrointestinal cancer, especially a colorectalcancer; or a tumor of the genitourinary tract, especially a prostatecancer, especially where such tumor is metastatic, wherein CHDMAPP isadministered between once-weekly and eight-weekly to a warm-bloodedanimal in a dose that is between the Efficient Concentration valuegiving half the maximum effect (EC50) and the Efficient Concentrationvalue giving the maximal effect (EC100), or that is between 110% and200% of the EC50, or preferably at least 110%, 120%, 130%, 150%, 160%,175% or 200% of the EC50 value.

Further aspects and advantages of the present invention will bedisclosed in the following experimental section, which should beregarded as illustrative and not limiting the scope of the presentapplication. A number of references are cited in the presentspecification; each of these cited references is incorporated herein byreference.

EXAMLES Example 1

Synthesis of BrHPP

All glassware and equipment were dried for several hours prior to use.Unless otherwise stated, the reagents and starting material were fromFluka. Trisodium (R,S)-3 bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP)was produced as white amorphous powder by the following procedure. Tosylchloride (4.8 g, 25 mmol) and 4-(N,N-dimethylamino-) pyridine (3.4 g,27.5 mmol; Aldrich) were mixed under magnetic stirring with 90 ml ofanhydrous dichloromethane in a 250-ml three-necked flask cooled in anice bath. A solution of 3-methyl-3-butene-1-ol (2.2 g, 25 mmol) in about10 ml of anhydrous dichloromethane was then slowly introduced with asyringe through a septum in the flask, and the ice bath was thenremoved. The reaction was monitored by silica gel TLC (pentane/ethylacetate, 85:15 (v/v)). After 2 h with constant stirring, the mixture wasprecipitated by dilution into 1 liter of hexane and filtered, and thefiltrate was concentrated under reduced pressure. Thisfiltration/suspension step was repeated using diethyl ether, and theresulting oil was purified by liquid chromatography on silica gel(pentane/ethyl acetate, 85:15 (v/v)), yielding a yellow oil of3-methyl-3-butene-1-yl-tosylate (5.6 g, 23.5 mmol, 94% yield) kept underdry N₂ at 4° C. (positive mode ESI-MS: m/z 241 [M+H]⁺; m/z 258 [M+NH₄]⁺;m/z 263 [M+Na]⁺; MS² of m/z 258: m/z 190 (C₅H₈ loss)).

Disodium dihydrogen pyrophosphate (51.5 mmol, 11.1 g) dissolved in 100ml of deionized water (adjusted to pH 9 with NH₄OH) was passed over acation exchange DOWEX 50WX8 (42 g, 200 meq of form H⁺) column and elutedwith 150 ml of deionized water (pH 9). The collected solution wasneutralized to pH 7.3 using tetra-n-butyl ammonium hydroxide andlyophilized. The resulting hygroscopic powder was solubilized withanhydrous acetonitrile and ether dried by repeated evaporation underreduced pressure. The resulting Tris (tetra-n-butyl ammonium)hydrogenopyrophosphate (97.5% purity by HPAEC; see below) was stored(concentration, ∞0.5 M) at −20° C. in anhydrous conditions undermolecular sieves. 100 ml of a solution containing 50 mmol of Tris(tetra-n-butyl ammonium) hydrogenopyrophosphate (0.5 M, 2.5 eq) inanhydrous acetonitrile under magnetic stirring in a 250-ml three-neckedflask cooled in an ice bath were slowly mixed with 20 mmol (4.8 g) of3-methyl-3-butene-1-yl-tosylate introduced via a septum with a syringe.After 20 min, the ice bath was withdrawn, and the reaction was leftunder agitation at room temperature for 24 h. The reaction was analyzedby HPAEC (see below), evaporated, and diluted into 50 ml of a mixturecomposed of a solution (98% volume) of ammonium hydrogenocarbonate (25mM) and 2-propanol (2 volume %). The resulting mixture was passed over acation exchange DOWEX 50WX8 (NH₄ ⁺, 750 meq) column formerlyequilibrated with 200 ml of the solution (98% volume) of ammoniumhydrogenocarbonate (25 mM) and 2-propanol (2 volume %). The column waseluted with 250 ml of the same solution at a slow flow and collected ina flask kept in an ice bath. The collected liquid was lyophilized, andthe resulting white powder was solubilized in 130 ml of ammoniumhydrogenocarbonate (0.1 M) and completed by 320 ml ofacetonitrile/2-propanol (v/v). After agitation, the white precipitate ofinorganic pyro- and mono-phosphates eliminated by centrifugation(2100×g, 10° C., 8 min). This procedure was repeated three times, thesupernatant was collected and dried, and the resulting oil was dilutedin 120 ml of water. Remainders of unreacted tosylates were extractedthree times by chloroform/methanol (7:3 (v/v)) in a separatory funnel,and the water phase was finally lyophilized. The resulting white powderwas again washed twice by acetonitrile/chloroform/methanol (50:35:15(v/v)) and dried under gentle N₂ flow. 11.25 mmol of pure3-methyl-3-butene-1-yl-pyrophosphate triammonium salt were obtained bythis procedure (75% yield) and were then dissolved in 200 ml of waterfor oxidation. For 6 mmol of 3-methyl-3-butene-1-yl-pyrophosphate, anaqueous solution of Br₂ (0.1 M) kept at 4° C. was added dropwise untilappearance of a persistent yellowish color, yielding after evaporation5.8 mmol (2.3 g) of an acidic solution (pH 2.1) of BrHPP, which wasimmediately neutralized by passing over DOWEX 50WX8-200 (NH₄ ⁺, 48 meq).The ammonium salt of BrHPP obtained after lyophilization was dissolvedin water and separated from bromides by passing through DionexOnGuard-Ag (2 meq/unit) cartridges and an on-line column of (100 meq, 21g) DOWEX 50WX8-200 (Na⁺) eluted by milli-Q water. Colorless stocksolutions of BrHPP (Na⁺) were filtered over Acrodisc 25 membranes of 0.2μM and kept as aliquots at −20° C.

HPLC—Final purification of BrHPP was achieved by HPLC (Spectra systemP1000 XR device) on an analytic Symmetry 5 μ C18 column (Waters) elutedat 1 ml/min and 20° C. with the ternary gradient indicated below.Upstream of detectors, a split of eluent distributes 190 μl/min in theonline MS detector (see below), and the remaining 810 μl/min was sent tothe Waters 996 photodiode array detector. Single wavelength detection atλ=226 nm was of 7 milliabsorbance units for 6 μg of BrHPP injected in 25μl (Rheodyne injector). The gradient program was as follows: solvent A,acetonitrile; solvent B, 50 mM ammonium acetate; solvent C, water; 0-7min, 5% B in C; 7.1-11 min, 100% C; 12-15 min, 100% A; 15-17 min, 100%C.

Example 2 Synthesis of HDMAPP using the method of Hecht et al (2002)(E)41Chloro-2-methylbut-2-en-1-ol

TiCl4 (285 mg, 1.5 mmol 164.5 μL) is dissolved in 3 mL of dry CH2CL2under N2. The solution is cooled to −80 to −90C, and a solution of 84 mgof commercially available 2-methyl-2-cinyloxirane (98.2 μL, 1 mmol) in0.4 mL of CH2CL2 is added in dropes with stirring. After 90 min. thereaction mixture is quenched by adding 5 mL of IN HCl. After warming toroom temperature, the phases are separated and the aqueous layer isextracted four times with 20 mL of diethyl ether. The combined organicphases are dried over MgSO4. Evaporation of the solvent and purificationby flash chromatography (pentanes/diethyl ether 1:1 v/v) affords 93 mgof pure product.

(E)-1-Hydroxy-2-methylbut-2-enyl 4-diphosphate from(E)-4-Chloro-2-methylbut-2-en-1-ol

A solution containing 227 mg (0.25 mmol) of tris (tetra-n-butylammonium)hydrogen pyrophosphate in 300 μL of MeCN is added slowly at roomtemperature to a solution of (E)-4-Chloro-2-methylbut-2-en-1-ol (25 mg,0.21 mmol) in 250 μL of MeCN affording an orange-red solution. After 2h, the solvent is removed under reduced pressure. The orange-colored oilis dissolved in 3 mL of H2O, and the solution is passed through a columnof DOWEX 50 WX8 (1×4 cm, NH4+ form) that has been equilibrated with 20mL of 25 mM NH4HCO3. The column is developed with 20 mL of 25 mMNH4HCO3. Fractions are combined and lyophilized to yield 0.19 mmol ofpure product (90%).

Example 3 BrHPP Non-GLP Studies

3.1. Material and Methods

3.1.1. Animals

Group 1: 5 purpose bred healthy male cynomolgus monkeys (M.fascicularis), supplied by C. R. P.

Le Vallon, Ferney S. E., Mahebourg, Mauritius. At the beginning of thestudy, body weights range from 3.7 to 4.6 kg.

Group 2: 10 purpose bred healthy cynomolgus monkeys (5 males and 5females), supplied by C. R. P. Le Vallon. At the beginning of the study,body weights range from 1.8 to 3.5 kg and ages from 2 to 3 years.

Husbandry conditions conformed to the European requirements, comprisingmonitored temperature, humidity, air change and lighting cycle. Group 1animals were housed in Biomatech (Chasse sur Rhône, France), and group 2animals were housed in MDS, (Les Oncins, France).

All experiments were subjected to local ethical committee beforeprocessing.

3.1.2. Phosphoantigens

The synthesis and characterization of trisodium(R,S)-3-(bromomethyl)-3-butanol-1-yl-diphosphate BrHPP has beendescribed previously (see above, as carried out in Espinosa, 2001). Thelot used for the experiments described here was manufactured andcharacterized under GMP conditions by PCAS-SELOC (Limay, France).Sterilization and clinical unit preparation was conducted under GLP byAXCELL BIOTECHNOLOGIES (Saint-Genis 1'Argentière, France). Titration ofBrHPP in sterile aqueous solution was achieved by High PerformanceAnion-Exchange Chromatography with conductimetric detection (DIONEXDX600 system).

3.1.3. Drug Administration and Blood Sampling

Group 1: animals were anaesthetised with intra-muscular injection of 6mg/kg ZoletilND 100 (Tiletamine-Zolazepam, Virbac, Carros, France)before any injection or blood taking.

Group 2: injections and blood taking were performed on manuallyrestrained non-anaesthetised animals.

4-day BrHPP alone treatment (group 1): 2 animals received 1 mg/kg ofBrHPP in 10 ml saline on day 0 (microflex infusion set introduced intothe external saphenous vein) and then 4 mg/kg, 16 mg/kg and 32 mg/kg in20 ml saline on days 1, 2 and 3 by the same way. (Duration of infusion:10 to 15 min).

BrHPP/IL2 co-treatments (group 1): 5 animals received either 20 mg/kgonce or 4 mg/kg (16.7 mg/kg or 3.3 mg/kg of BrHPP anionic form) 5 timesdaily of BrHPP in final 50 ml saline by the same way as above (durationof infusion: 30 min). IL2 (18 million UI per vial, Proleukin®, Chiron,US) was resuspended in 1 ml sterile water and diluted to 10 ml with 4%HSA for a final concentration of 1.8 million UI/ml. In a first cycle oftreatment, all animals received the same dose of IL2 consisting of 5days of twice daily injections of 0.9 million units IL2. In a secondcycle of treatment, animals received subcutaneously the following IL2treatment: 0.15 million units twice daily for 9 days (animal Z059), 0.3million units twice daily for 5 days (Z135), 0.9 million units twicedaily for 5 days (animal Z714) or 9 days (animal X973).

A single animal received 80 mg/kg (66.6 mg/kg anionic form) BrHPP+9 daysof IL2 co-treatment consisting of a single daily subcutaneous injectionof 0.6 million units.

BrHPP/IL2 co-treatments (group 2): BrHPP was diluted in saline to theappropriate final concentration (depending on the dose to inject and onthe last recorded body weight) so as to inject always approximately 50mlin 30min (microflex infusion set introduced into cephalic or externalsaphenous vein). All animals received 0.6 million IU IL2 per day for 7days. IL2 was administered subcutaneously as 2 separate injections of0.3 million IU IL2 in sterile water, 8-hour apart. Control animalsreceived IL2 only.

Twice weekly blood samples (1 to 4 ml) were withdrawn from femoralvessels/artery into EDTA containing tubes. Tubes were shipped overnightat room temperature (RT) before flow cytometry analyses.

3.1.4. Flow Cytometry

Peripheral γδ lymphocytes were analysed twice weekly by flow cytometryon total monkey blood, after double staining with anti-CD3-PE antibodyand anti-Vgamma9-FITC antibodies and/or anti Vd2 antibodies (CD3-PE:SP34 clone, BD Biosciences Pharmingen, Le Pont de Claix, France). AntiVgamma 9, clone 7B6 is a monoclonal raised to human Vgamma 9 but thatcross-reacts with cynomolgus cells. It was purified by affinitychromatography on protein A and coupled to FITC as previously described.We checked that this antibody stained most of Vd2 positive cells,stained by commercial (Endogen, Woburn, Mass.) TCR2732 clone (aspreviously described for other Vg9 antibodies in Rhesus monkey by Shen,2002, data not shown).

Briefly, 50 μl monkey blood is incubated 15 min at RT with 5 μlanti-CD3-PE and 6 μl anti-delta2-FITC or 10 μl anti-gamma9-FITCantibodies. Antibodies are washed with 3ml 1× PBS, centrifuged for 4 minat 1300 rpm at RT and supernatant is discarded. Red cells are lysed withthe OptiLyse C reagent (Immunotech-Beckman-Coulter, Marseilles, France)according to the manufacturer's instructions. At the final step, stainedwhite blood cells are recovered by centrifugation and resuspended in 300μl PBS+0.2% PFA. Immediately before analysis, 50 μl calibrated FlowCount™ Fluorospheres (Immunotech-Beckman-Coulter, Marseilles, France)are added to the cells for absolute number counting of the populationsof interest.

For group 2, lymphocyte subsets were also analysed in parallel by dualcolor flow cytometry with CD20-FITC (2H7 clone); CD3-PE (SP34 clone);CD4-FITC (M-T477 clone); CD8-FITC (SK1 clone) (all purchased from BDBiosciences, Le Pont de Claix, France).

Flow cytometry was performed on a Epics XL-MCL apparatus(Beckman-Coulter, Roissy, France) with the Expo32 software.

3.1.5. Cytokine Detection

Serum cytokines (TNFa and INFg) were detected and quantified with theBIOSOURCE Cytoscreen™ ELISA monkey TNFa and Cytoscreen™ ELISA monkeyINFg respectively (purchased from CliniSciences, Montrouge, France)according to the manufacturer's instructions.

3.1.6. Haematology and Serum Clinical Chemistry

Classical blood parameters follow-up (red blood cell, platelets totaland differential white blood cells counts, haemoglobin, mean corpuscularhaemoglobin, mean corpuscular haemoglobin concentration) were performedat the sites of monkey handling, just before and after eachadministration and twice weekly.

On all animals in group 2, 48 or 72 hours after each injection, 16 bloodchemistry parameters were measured (sodium, potassium, chloride,calcium, inorganic phosphorus, glucose, urea, total cholesterol, totalbilirubin, total protein, albumin, globulin, creatinin, alkalinephosphatase, aspartate aminotransferase and alkaline aminotransferase).

3.1.7. Vital Parameters Follow Up

The animals were observed daily and during and after each injection forany change in vital and clinical parameters (general behaviour, skin,hair, respiratory system, central nervous system). Animals wereregularly weighed, every 3 days (group 1) or weekly (group 2). Bodytemperature (on vigil animals of group 2) was measured before and at theend of each BrHPP/IL2 (or IL2 alone) infusion and once daily during the5 days following administration. Heart rate and blood pressure wererecorded for all animals in group 2, before and at the end of eachadministration. All animals were observed at least twice daily for signsof morbidity/mortality.

3.1.8. Biopsy Preparations

5 animals in group 2 were sacrificed 9 days after the secondadministration, by intravenous injection of sodium pentobarbitone,exsanguinated, and were submitted to full necropsy procedures for organtoxicology assessment of the drug. Samples of organs were weighed andcollected in RPMI medium (Gibco-BRL—Life Science) for furtherprocessing.

Lymphoid organ samples (thymus, tonsils, bone marrow, mesenteric,inguinal and tracheo-bronchial lymph nodes) were carefully mechanicallydissociated with sterile syringe plungers, washed several times in RPMImedium and filtered twice through nylon membranes (Scrynel NYHC 100 μmnylon, purchased from VWR International, France). Cells were then doublestained with anti-CD3-PE and anti-gamma9-FTIC antibodies and analysed byflow cytometry as described above.

3.2. Results

3.2.1. BrHPP Alone Does Not Induce Reproducible Expansion of g9d2 Cellsin vivo

We tested first if BrHPP injection alone was able to support activationof g9d2 positive T cells in vivo.

In a first series of experiment, 4 monkeys were treated by five dailyinjection of 0.2 mg/kg (0.17 mg/kg anionic form) of BrHPP, and 4 animalswere treated with saline. Injection of this dose of BrHPP did not resultin any toxicity in the treated non human primates as assessed by vitalsigns or rectal temperature. Follow up of Vg9Vd2 T cells showed a slightincrease in % of Vg9Vd2 in two treated animals and none in the placebogroup but this expansion was not significant (data not shown).

To test if higher doses of the molecule could induce a more consistentactivation or expansion, another two animals received four dailyinjections with increasing dosing of BrHPP (1 mg/kg, 4 mg/kg, 16 mg/kgand 32 mg/kg, or the equivalent respective doses of 0.83 mg/kg, 3.33mg/kg, 13.33 mg/kg and 26.6 mg/kg anionic form BrHPP).

Again no toxicity nor fever was observed in the two treated animals.Follow up of Vg9Vd2 reavealed a decrease at day two post firstinjection, but the two animals returned to basal level at day 9 (FIG.1). Survey-of CD25 and CD69 did not give significant results, but thesemarkers may not be optimal for study of non human primate cells.

At least two possibities can explain the lack of objective response ofVg9Vd2 cells upon BrHPP treatment i) as the BrHPP is a small moleculecontaining pyrophosphate, it may be excreted or degraded so rapidly thatit does not allow sufficient contact with the target cells to allowactivation of the cells ii) Expansion of g9d2 cells in vivo requires, asin vitro, the presence of cytokine, particularly IL2.

Although we do not have at present an analytical method sensitive enoughto follow the molecule in vivo, it was likely that the serumconcentration in vivo would be sufficient to trigger Vg9Vd2 cells. Invitro, we and others (Lang, 1995) have shown that the activation ofVg9Vd2 cells occurs within minutes after the addition of the molecule.Two mechanisms may prevent the molecule to be active in vivo i) thelikely rapid renal clearance of such a small molecule and ii) adegradation process in vivo. The study of the half life of the moleculein primate sera showed that the half life of the molecule at 37° C. isabout one hour. The main mechanism of degradation of the molecule is theremoval of the first phosphate, rendering the molecule biologicallyinactive. Nevertheless, the concentration of the molecule, injected byintravenous route in relatively high amount, should be maintained beyondthe 20 nM EC50 for a significant time, if no additional, blood cell ororgan related degradation process occurs in vivo.

To try to reveal the activation of the cells, we tested the concomitantinjection of low doses of rhIL2 with BrHPP.

3.2.2. BrHPP+IL2 Induces Reproducible, Transient Expansion of Vg9Vd2Cells in vivo in Cynomolgus.

4 animals and one control (group 1 animals) were treated with thecombination of BrHPP or saline with low doses of rhIL2. This group ofanimals was composed of the two previous animals that did not show anyaugmentation of Vg9Vd2 upon injection of BrHPP alone and three newanimals. Among the four BrHPP treated animals 2 animals underwent 5daily injection of 4 mg/kg of BrHPP, and two animals underwent a singleinjection of 20 mg/kg (16.7 mg/kg anionic form). The BrHPP treatedanimals and control were administered the same rhIL2 regimen consistingof 0.9 million units twice daily by subcutaneous injection for 5 days.The control animal was treated first with saline and IL2, and then 14days later with a single shot of BrHPP and a second course of 5 daysIL2.

Haematological follow up of the animals revealed increase of lymphocytecounts by 2 to 4 in the BrHPP treated animals and control as well,consistent with the [IL2 administration.

A specific expansion both in percent increase among CD3 positive cellsand absolute numbers of Vg9Vd2 was observed in all BrHPP treated animals(FIG. 2). This increase was transient, as the expansion is seen at day 7with already a slight increase at day 3, and levels being back to aroundbefore treatment level at day 10. The control animal treated with IL2alone, showed a slight increase in absolute numbers but no increase inpercentage, suggesting that peripheral Vg9Vd2, as other lymphocytesubset, slightly increase upon IL2 administration. Administration ofBrHPP on the control animal, after this cycle of IL2 alone, results inan expansion consistent with that of treated animals, thereforevalidating this animal as a control.

Absolute increase of Vg9Vd2 was higher in the two animals receiving asingle dose of BRHPP (×30 and ×40) as compared to animals receiving thesame dose but split in 5 daily injections (×8 and ×18) (FIG. 1c). Thismay be due either to the deleterious effect of multiple injection orlower individual dose injected. As single injection was at least asefficient and more practical, next experiments were performed withsingle injection of the product.

3.2.3. Dose Range Effect of BrHPP in Cynomolgus Monkey

To evaluate the dose range effect of the molecule, a new experiment wasset on 10 new animals (group 2 animals). Subgroups of two animals, onemale and one female, were treated with increasing doses of BrHPP (0,0.2, 4, 20, 80 mg/kg), and co-treated with IL2 for 7 days.

Again, animals treated with EL2 alone did experience a slight increaseof Vg9Vd2 cells, of the same order of magnitude as compared to otherlymphocyte subset.

0.2 mg/kg dose was undistinguishable from the control animals, both interms of % and absolute numbers of Vg9Vd2. A dose range effect wasobserved both in % and absolute numbers of Vg9Vd2 from 4 to 80 mg/kg(FIG. 3 a) at day 7, without apparently reaching a plateau. Again % andabsolute counts came rapidly back to pre-treatment level.

As a plateau was not reached during this treatment, same animals weretreated a second time 21 days post first injection, after Vg9Vd2 cameback to pretreatment level, with doses ranging from 20 mg/kg, 80 mg/kg,120 mg/kg and 160 mg /kg. To minimize potential effect of the firsttreatment on the second treatment, the new doses (120 and 160 mg/kg)were given to animals that seldom responded to the first treatment (ieanimals treated with 0.2, and 4 mg/kg during the first course oftreatment, FIG. 3 b).

The time line of the proliferation after this second injection was aboutthe same as described before. It should be noted that at the highestconcentration, the level of circulating Vg9Vd2 reached about 80% ofcirculating CD3 positive cells (FIG. 3 b), with absolute numbersreaching a mean of 10,000 Vg9Vd2 cells/mm3. The numbers and percentageof Vg9Vd2 declined after the peak between day 5 and day 7 , although thetime to go back to pre-injection level appears slower for the twohighest doses.

Pooled data from injection 1 and injection 2 gave a clear dose rangeeffect both in terms of percentage and absolute numbers of Vg9Vd2 cellsat the peak of response (FIG. 3 c and 3 d). This dose range effect isobserved despite some individual heterogeneity, particularly in the 120mg/kg group, as demonstrated by the error bars, with a no effect dosesituated between 0.2 and 4 mg/kg, still higher doses would have beennecessary to establish a plateau.

3.2.4. Influence of IL2 Schedule

5 animals from group 1 were submitted to injections of BrHPP followed bydifferent doses and time schedule of IL2. As shown in FIG. 6, even dosesas low as 0.15 million units twice daily are sufficient to supportexpansion of Vγ9Vδ2 T cells, apparently equivalent to higher doses (0.3and 0.9 million units twice daily). The length of the Vγ9Vδ2 expansiondoes not seem to increase when IL2 treatment is given for more than fivedays, although more animals should be tested to clearly establish it.Interestingly, an animal subjected to 0.6 million units subcutaneousinjection of IL2 as a single daily injection also developed an expansionof Vγ9Vδ2 cells (data not shown).

3.2.5. Phosphoantigen+IL2 Induced Expansion can be Reproduced at LeastFive Times Successively

A memory type response to a recall injection of phosphoantigencontaining preparation (ie BCG) was recently suggested in a primatemodel. We tested if such a response could be obtained with a purepreparation of synthetic ligand, and IL2 cotreatment.

Animals from group 1 were treated a second time with a single shot of 20mg/kg and 7 days IL2 and Vg9Vd2 increase was monitored by flowcytometry. Fold increase obtained with the two injections is representedin FIG. 4 a. The timing of the amplification of Vg9Vd2 was the same asin the first injection. The two animals that were treated with a singleshot of BrHPP during the first injection showed a slightly reducedamplification rate in the second injection. The two animals that weretreated with 5 daily 4 mg/kg of BrHPP injections showed a slightlyhigher amplification rate at the second injection, most probablyreflecting the better effectiveness of single injections overfractionated injections as discussed before.

As mentioned before, animals of group 2 underwent two successiveinjections during the dose range assay. Interestingly, the two animalsthat were treated first with 80 mg/kg, and then with 20 mg/kg showed adecreased expansion, as compared to all animals tested with a firstinjection of BrHPP at 20 mg/kg (FIG. 4 b). Animals treated first with 20mg and then with 80 mg showed expansions comparable to animals treatedwith 80 mg/kg at the first injection. Taken together, these resultssuggest that response of Vg9Vd2 to BrHPP+IL2 expansion can be repeatedtwice, although may be with lower expansion rate.

To get further insight into the possible influence of successiveinjections on the Vg9Vd2 response, 5 animals from group 2 (one animalper subgroup) were treated with a third cycle of BrHPP injection 21 daysafter the second injection and a fourth cycle after another 21 days.

Again the kinetic of Vγ9Vδ2 expansion was the same as describedpreviously, with a peak between day 5 and day 9. As shown in FIG. 3c,one animal gave a high increase at the third injection (×28).Interestingly, this animal was the only one which did not come back tothe basal level after the second injection of 120 mg/kg (animal 2034,FIG. 3B). The control animal which received only IL2 in the first twocourses of treatment gave an increase of around 40, compatible with thevalues obtained in four animals treated with this dose in the first twotreatment cycles (FIG. 4 b). The three other animals gave amplificationlower than 40, lower than amplification in animals treated with the samedose at injection 1 or 2.

The five animals treated with a fourth injection of 80 mg/kg BrHPP andIL2 gave a detectable increase of Vg9Vd2 although lower than thatobtained in the third injection (FIG. 4 b).

In conclusion, it is shown that induction of proliferation of Vg9Vd2 byBrHPP +IL2 co-treatment can be reproduced several times. In theseexperiments, we could achieve expansion 4 successive times, withinjections of BrHPP separated by 20 days or higher.

3.2.6. Short Term Production of Cytokine by g9d2 T Cells in vivo UponChallenge with Phosphoantigens

Vg9Vd2 cells are known producers of TNFa and IFNg in vitro uponphosphoantigen challenge. In order to evaluate if these cells could be asource of these cytokines in vivo, sera of some animals was collectedshortly after BrHPP injection.

Samples of sera were collected from group 2 animals during the firstdose range assay (0 to 80 mg/kg) just before injection then 1 and 4hours post injection, and assayed by ELISA specific for TNFa and IFNg.

Assessment of IFNγ did not give significant results in all treatedanimals.

INFα was detectable in the sera of animals treated at the highest dose(80 mg/kg) one hour after injection of BrHPP (FIG. 5 a). The serum levelof TNFa rapidly decreased as it was not further detectable 4 hours postBrHPP injection and remains undetectable during the increase of Vg9Vd2in blood. This suggests that TNFa is rapidly produced fromintracellular, pre-formed pool by Vg9Vd2, and then is seldom producedafter the initial activation by the drug.

To test the capacity of cells to produce cytokines after expansion withthe drug, two animals of group 2 were injected a fifth time at 80 mg/kgwith BrHPP (without IL2) at day 7 post fourth 80 mg/kg injection, wherethe level of Vg9Vd2 was at the peak. As shown in FIG. 5B, both TNFa andIFNg was detectable in the sera of treated animals. Production of TNFafollowed the same kinetic as compared to the first experiment, with alevel becoming undetectable at 4 hours post injection. On the reverse,IFNg became detectable at one hour post injection, and increased at 4hours post injection, suggesting a slower, but more sustained productionof this cytokine.

3.2.7. Absence of Toxicity of Injection of BrHPP Alone or in Associationwith IL2

No alteration of clinical or blood chemical parameters (see material andmethods) were seen in any of the animals treated either with BrHPP aloneor in association with IL2. From an haematologic point of view, in allanimals treated with IL2, a transient increase (2 to 5 times) inlymphocytes was observed. In animals treated with the highest dose ofBrHPP, a lymphocytosis was observed, corresponding to the peak of Vg9Vd2in the periphery.

It should be noted that rectal temperature was not significantlyaffected in any animal treated, despite the presence of detectable levelof TNFα and IFNγ inflammatory cytokines in sera of some treated animals.

Example 4 Phosphostim (BrHPP) I.V. Pharmaco-Dynamic Study in CynomolgusMonkeys

The objective of this GLP study was to explore further thepharmaco-dynamic properties of Phosphostim (BrHPP, 200 mg; GMP batch)alone and in combination with IL-2 following several i.v.administrations to cynomolgus monkeys. In particular, this study wasplanned to evaluate:

-   -   A dose-effect relationship between Phosphostim injection and γδ        T cell peripheral amplification;    -   The pharmaco-dynamic effect of a second, third and up to fifth        administration of Phosphostim in male and female animals;    -   The functionality of in vivo induced γδ T cells, by dosing        systemic cytokines after Phosphostim treatment;    -   The effect of treatment with IL-2 alone and to validate the        selected dose of 0.6 million IU/day/animal.

Phosphostim was administered i.v., as slow infusions (50 ml in 30minutes), at various dose levels to five groups of two animals (onemale+one female). Males received two successive injections of BrHPP (Day0 and Day 22) before sacrifice and females four injections (Days 0, 22,52 and 84). Two females, selected upon their level of response to the4^(th) treatment, received a supplementary injection of BrHPP alone atDay 91, during γδ cell peripheral increase, for systemic cytokinedosages.

The administration schedule of the test product can be summarized asfollows in Table 1: TABLE 1 BrHPP Dose Levels (mg/kg)*** Day 0 Day 22Day 52 Day 84 Day 91 1^(st) 2^(nd) 3^(rd) 4^(th) 5^(th) Group #injection injection injection injection injection^(¥) 1  0*   0* 80**80** 80*** 2   0.2 160 80** 80** 80*** 3  4 120 80** 80** — 4 20  8080** 80** — 5 80  50 80** 80** —*administration of Ringer lactate physiological solution alone as acontrol**females only***selected females, upon their level of response to the 4^(th)injection^(¥)without IL-2 co-treatment

The dose levels in Table 1 are can be converted to BrHPP pure (anionic)form equivalent by multiplying each dose by 0.6. Thus, the doses of 0.2,4, 20, 50, 80, 120 and 160 mg/kg in Table 1 are equivalent respectivelyto 0.12, 2.4, 12, 30, 48, 72 and 96 mg/kg of anionic form BrHPP.

IL-2 was administered s.c. at the following frequency: from Day 0 to Day6 and from Day 22 to Day 28 for all animals; from Day 52 to 58 and fromDay 84 to Day 90 for all the females. IL-2 was administered as twoseparate s.c. injections approximately 8 hours apart of 0.3 million IU,corresponding to 0.6 million IU/day/animal.

Results

The first and second administrations of Phosphostim and IL-2 resulted ina clear dose-related elevation of peripheral Vγ9V52 T cells at Day 7,which is represented in FIG. 7.

The first slightly efficient tested dose was 4 mg/kg (2.4 mg/kg anionicform BrHPP), 20 mg/kg (12 mg/kg anionic form BrHPP) inducing 20-fold γδcell number increase. The estimated EC50 value of BrHPP in vivo wasaround 120 mg/kg (72 mg/kg anionic form BrHPP) and induced a 100-foldincrease in circulating γδ cell count. At the highest tested dose,circulating Vγ9Vδ2 T cells were found 200-fold more numerous than beforetreatment and represented the majority of peripheral lymphocytes.

This study confirmed that IL-2 alone induced no specific amplificationof γδ T cells in vivo.

The effects of Phosphostim treatment on the production of cytokines(INFγ and TNFα) production in the serum was studied twice:

-   -   after the first infusion, in all treated animals: a significant        production of systemic TNFα (serum concentrations around 60 and        120 pg/ml) was evidenced in both animals having received 80        mg/kg, 1 hour after BrHPP injection;    -   in two females (F2032 & F2034), which received 80 mg/kg (48        mg/kg anionic form BrHPP) (without IL-2) during the peak (Day 7)        of the 4^(th) injection. Serum TNFα and INFγ concentration        evolutions for both animals are shown on the following curves        (see FIG. 8).

Thus, Vγ9Vδ2 T cells amplified in vivo upon BrHPP/IL-2 co-treatment alsoproduce detectable amounts of systemic cytokines.

Example 5 Supplementary Pharmaco-Dynamics Studies in the Primate

We have thus performed other non-GLP pharmaco-dynamics studies in malecynomolgus monkeys in order to further document the features of in vivoγδ T cells response to consecutive injections of BrHPP, varying eitherthe dose or the time-laps between injections. We have demonstrated that,in males:

-   -   at least 3 successive injections, 8 weeks apart, of either 20 or        80 mg/kg (12 or 48 mg/kg anionic form equilvalent) BrHPP induce        significant increases of γδ cell rate and absolute count in the        periphery;    -   at least 4 successive injections, 4 weeks apart, of 20 mg/kg (12        mg/kg anionic form equilvalent) BrHPP also induce detectable        increases of γδ cell rate and absolute count.

Thus we confirm that under some circumstances, at least four successiveperipheral amplifications of γδ T cells induced by BrHPP/AL2co-treatment can be obtained, indifferently in male and femalecynomolgus monkeys.

Example 6 Two-Phase Repeated Dose Toxicity Studies in Cynomolgus MonkeysFollowing I.V. Administration of Phosphostim and BrHPP

The objective of the first phase of this non GLP study was to determineBrHPP Maximum Tolerated Dose (MTD) in a group of two cynomolgus monkeys(one male, one female) by escalating doses. The second objective of thisstudy was to characterize the toxicity of daily i.v. administration ofPhosphostim for two weeks in two cynomolgus monkeys (one male, onefemale) at the MTD determined during the first phase of the study. Theanimals were treated at increasing dose-levels every 3 or 4 days(phase 1) and then at the estimated Maximum Tolerated Dose (MTD) dailyfor 2 weeks (phase 2).

A total of four cynomolgus monkeys (two males and two females) weredivided into 2 groups and treated with BrHPP, by intravenousadministration over a 1-hour infusion period as follows:

Phase 1 (Escalating Dose):

Group 1 (one male and one female) were administered BrHPP and/orPhosphostim (BrHPP, 200 mg) i.v. at increasing dose-levels (160, 400,600, 900 and 1200 mg/kg) every 3 or 4 days.

Anionic form BrHPP equilvalent to these doses are respectively: 96, 240,360, 540 and 720 mg/kg. BrHPP was given as a solution afterreconstitution in water for injection finally diluted in

Ringer lactate under a dose volume of 4, 10 or 15 mL/kg.

Phase 2 (Fixed Dose):

Group 2 (one male and one female) were administered BrBPP i.v. daily at900 mg/kg/day (540 mg/kg/day in anionic form BrHPP equivalent) for 2weeks.

The animals were checked daily for mortality and clinical signs. Bodyweight was recorded pre-study and on the day of each administration inphase 1 or twice weekly throughout phase 2, and including on the day ofnecropsy for phase 2. Food consumption was estimated daily, starting atleast 7 days pre-phase. Hematological, blood biochemical and/orperipheral blood lymphocyte subset analysis investigations wereperformed pre-study, on the day of each administration in phase 1, onday 7 in phase 2 and at the end of both phases. Electrocardiographic andblood pressure recordings were performed pre-study, on the day of eachadministration in phase 1 and on day 1 and at the end of treatment inphase 2. Blood, for determination of plasma levels of the test item, wassampled on days 1 and 14 in phase 2. On completion of each phase,animals were submitted to a complete macroscopic post-mortem examinationand specified tissues preserved. In phase 2, selected body organs wereweighed and microscopic examination of selected tissues was performedfor all animals.

Results

No unscheduled death occurred and no relevant clinical signs were notedduring the treatment phases. BrBPP treatment had no effect on the bodyweight evolution of animals in either phase and food consumption wasunaffected.

BrHPP did not affect blood pressure in either phase. No treatmentrelated hematological or blood chemistry changes were noted in phase 1.In Phase 2, increased lymphocyte count was noted in both animals, whichmay be attributed to the pharmacological activity of BrHPP.

A slightly lower absolute thymus weight was noted in the female.

No relevant findings were found either during Phase 1 or Phase 2 aftermacroscopic post-mortem examination. Lymphoid depletion was seen in thethymus and mesenteric lymph node of the female, whereas the oppositetrend (increase of follicular germinal center in the mesenteric lymphnode) was noted in the male in phase 2.

Example 7 Two Week Repeated Dose Toxicity Study in Rats Following I.V.Administration of Phosphostim

The objective of this GLP study was to evaluate the potential toxicityof BrHPP following daily i.v. administration in rats for two weeks. 116Sprague-Dawley rats were allocated into four groups: one control group(Group 1) with 10 males and 10 females and three treatment groups(Groups 2 to 4) with 10 males and 10 females. Group 2, 3 and 4 wereadministered respectively 80, 150 and 300 mg/kg/day (respectively 48, 90and 180 mg/kg in anionic form equivalent) of BrHPP. Each treatment groupalso had a satellite group of 6 females and 6 males. Satellite animalswere allocated for toxico-kinetics purposes.

BrHPP was administered daily by slow intravenous injection (0.4 mL/min)as a solution in sterile water for injection and Ringer lactate at thedose levels of 80, 150 or 300 mg/kg/day. The control group received thevehicle (together with 1% sodium to reach an osmolarity similar to thehigh-dose group dosage forms) under the same experimental conditions. Aconstant dosage-volume of 5 mL/kg was used.

The animals were checked daily for mortality and clinical signs. Bodyweight and food consumption were recorded twice a week. Ophthalmologicexamination was performed before and at the end of the treatment period.Blood samples for determination of levels of BrHPP were taken fromsatellite animals on Day 1 and at the end of the treatment period.Vaginal lavage was performed in females during pre-treatment and at theend of the treatment period for monitoring of estrous cycle.Hematological, blood biochemical and urinalysis investigations wereperformed on all principal animals at the end of week 2. On completionof the study, all animals were killed and subjected to a macroscopicpost mortem examination and specified organs were weighed and preserved.Microscopic examination was performed on selected tissues from animalsof the control and the 300 mg/kg/day groups.

Results

No unscheduled death occurred during the study. Vocalization duringdosing was noted on many occasions in all animals given 150 and 300mg/kg/day (respectively 90 and 180 mg/kg in anionic form equivalent).Dose-related higher mean body weight gain was noted in females treatedwith BrHPP.

Similar mean food consumption was noted between control andBrHPP-treated animals. No relevant ophthalmologic findings were noted inanimals given 300 mg/kg/day (180 mg/kg in anionic form equivalent) atthe end of the treatment period. No changes of toxicologicalsignificance were noted in any hematological, biochemical or urinaryparameter. No relevant differences in organ weights from controls werenoted in any BrHPP-treated group at the end of the treatment period. Norelevant findings were noted in any BrHPP-treated group at the end ofthe treatment period from the macroscopic post-mortem examination. Uponmicroscopic examination, slight changes were observed in the tail veinof both control and BrHPP-treated animals, and were attributed to themechanical injury due to daily intravenous administrations. No relevantfindings were seen in other organs and tissues of animals given 300mg/kg/day. Additional results regarding blood levels of BrHPP,monitoring of the estrous cycle and toxico-kinetics are currently beinganalyzed.

Conclusion

BrHPP No Observed Adverse Effect Level (NOAEL) was considered to be 300mg/kg/day (180 mg/kg in anionic form equivalent), the highest testeddose under these study experimental conditions.

Example 8 Analysis of BrHPP-Amplified γδ T Cells Cytotoxicity TowardsAutologous Primary Tumor Cells of RCC Patients

The aim of the present study was

-   -   to establish primary normal and tumor renal cells from RCC        patients    -   to investigate the effect of the BrHPP on PBMCs of these        patients    -   to investigate the lytic potential of expanded Vγ9Vδ2 T cells        against primary normal and tumor renal cells in an autologous        setting. This cytotoxic activity was compared with other        autologous effectors cells, like LAK cells (Lymphokine-Activated        Killer cells) for example.        8.1 Material and Methods        Patients

All mRCC patients (N=12) included in this study presented a renal clearcells carcinoma and underwent partial or total nephrectomy. None of thepatients has received any previous treatment. Seven out of the 12patients were not included after the initial phase of testing for themain part of the study. Informed consent was obtained from the mRCCpatients.

Expansion of Peripheral Blood Gamma Delta T Cells

Blood samples from 12 RCC patients (50 ml) were collected just before orno more than 2 months after the nephrectomy. PBMCs were isolated bycentrifugation on Ficoll-Hypaque density gradient (AmershamBiosciences). PBMCs from one healthy volunteer was used as control.

Ten million PBMCs were cultured at 2×10⁶/ml in 24 well plate in RPMI1640 supplemented with 10% v/v fetal calf serum (Fetal Clone irradiated,Hyclone).

Polyclonal Vγ9Vδ2 T cell lines were specifically expanded in presence of3 μM BrHPP molecule (batch INPA-0214) and 100 IU/ml IL2 during 15 days.

Every three days, the volume corresponding to half culture medium wasreplaced by fresh medium containing only 200 IU/ml IL2

Obtention of LAks Cells

Five millions of PBMCs from mRCC patients were cultured in 6-well platein presence of 1000 UI/ml IL-2 for three days. Their cytotoxic activitywas assessed in a 4-hour ⁵¹Cr release assay in parallel with autologousexpanded Vγ9Vδ2 T cells to compare the cytotoxic efficiency of bothpopulations.

Establishment and Culture of Tumoral/Normal Cell Lines

The autologous primary tumor cell lines were derived from the tumorfragments by enzymatic digestion using Collagenase (300 U/ml),Deoxyribonuclase I (500 U/ml), Hyaluronidase (3000 U/ml) (SigmaAldrich). The same protocol was applied to a renal normal fragment,taken at distance from the tumor, to derive short-term normal renalcells.

These cells were cultured in Dulbecco modified Eagle medium supplementedwith 10% FCS and Ultroser (Gibco BRL, Scotland).

Cell Type Characterization

PBMCs from patients are either frozen or used directly in culture asmentioned above.

Before in vitro culture, phenotype of 2×10⁵ PBMCs from RCC patients wasanalyzed using the following combinations of fluorescein isothiocyanate(FITC), phycoeryibrin (PE) and phycoreythrin-cyanin 5 (PC5) conjugatedantibodies to determine the NK, αβ and γδ T cells population (allpurchased from Beckman-Coulter): CD3-PC5 /Vδ2-FITC /IgG1-PE, CD8-PE,CD56-PE. At day 15 of the culture, a triple staining was performed onthe expanded Vγ9Vδ2 T cells bulk The following antibodies combinationswere performed:

CD3-PC5/Vδ2-FITC/CD8-PE (IgG1), CD16-PE (IgG1), CD2-PE (IgG1), CD56-PE(IgG1), CD69-PE (IgG1), HLA-DR-PE (IgG1), CD45RO-PE (IgG1), NKG2D-PE(IgG1), NKG2A-PE (IgG2b), CD94-PE (IgG1), CD158a, b, e-PE (IgG1).

Background levels were measured using isotypic controls. Compensationwas set up with single stained samples, low forward scatter elementswere excluded from analysis and 10,000 events were collected andanalyzed using the Cell Quest software (Becton Dickinson).

Primary normal and tumor renal cells were phenotyped by indirect singlecolor fluorescence. Cells (2×10⁵) were incubated for 30 minutes at 4° C.with following antibodies:

anti-HLA-A2, anti-HLA-BC (clone B1.23.2), anti-HLA-ABC (clone W6/32),CD54, anti-MICA (clone BAM 195 provided by Prof. A. Moretta(Genova,Italy)), G250-FITC (provided by Dr. Hirsch F. Hôpital PaulBrousse, Paris), anti-human fibroblast (clone AS02 Dianova, Hamburg).Cells were washed twice with phosphate-buffered saline (PBS) and thenincubated for 20 minutes at 4° C. with phycoerythrin (PE)-conjugatedgoat anti-mouse Ig.

Assay for Cytolytic Activity

Expanded γδ effector T cells were tested for cytotoxicity againstautologous normal and tumor target cell lines, control sensitive targetcell line Daudi and control resistant target cell line Raji in 4 h ⁵¹Crrelease assay. LAK effector T cells for the same patient were includedin the assay to compare their lytic potential against the one of γδ Tcells.

-   -   Target cells were used in amounts of 2×10³ cells/well and        labeled with 100 μCi ⁵¹Cr for 60 minutes. Effector/Target (E/T)        ratio ranged from 30:1 to 3.75:1.    -   Specific lysis (expressed as percentage) was calculated using        the standard formula ((experimental-spontaneous        release/total-spontaneous release)×100). Data are the mean of        triplicate wells.        8.2 Results        Selective Expansion of TCR Vδ2-Bearing T Cells in PBMCs of        Patients with RCC

First, the ability of BrHPP to expand resting γδ T cells from peripheralblood of mRCC patients was compared to γδ T cells expansion of onehealthy volunteer. Seven patients could not be included in the studysince no blood has been collected (BAZ, DEN,GOU and SAN) or since thepathology is not a clear cell (or conventional cell) renal carcinoma(COU, FAB and ROU).

For the evaluable patients, PBMCs were stimulated as described inmaterials and methods. From twelve patients analyzed, seven of them(7/12 or 58%), namely BEL, FOUR, MOR, POU, QUI, VAG and ZEN presented aVγ9Vδ2 T cells amplification. These mRCC patients had from 1,6% to 4.2%γδ T cells at baseline that expanded from 73.3% to 94.8% afterstimulation with BrHPP (n=7).

Five patients, namely CHA, CHAR, PAS, SAU and SCH were not included inthe cytotoxicity assay since it was impossible to expand specificallytheir Vγ9Vδ2 population in vitro. These “BrHPP non responder patients”had from 0.6% to 3.8% γδ T cells at baseline that expanded from 4.7% to19.6% after stimulation (n=5).

When blood sample before nephrectomy was available, the experiment wasperformed with this sample. It has been checked that same amplificationresults could be obtained with a blood sample collected afternephrectomy (data not shown).

Cell Type Characterization of “BrHPP Responder Patients”

Effector Cells

Phenotypic analysis of expanded Vγ9Vδ2 T cells from mRCC patients thatrespond to BrHPP was carried out. Data from patients QUI and FOUR arenot shown for reasons detailed below. Most of these cells had thephenotype of Ag experienced cells (CD45RO+), expressed adhesionmolecules like LFA-1 or CD2 and expressed the costimulatory moleculeNKG2D. Some γδ T cells maintained an activated phenotype as confirmed bythe expression of CD69 and HLA-DR. Different subpopulations expressingeither CD8, CD56 or CD16 could be also identified. Whereas theinhibitory heterodimer complex CD94/NKG2A was expressed by almost halfof γδ T cells, receptors belonging to the Killer Ig-like Receptor family(CD158a; b; e) were expressed on very restricted subsets.

Target Cells

Phenotypic analysis of the tumor and normal renal cells from responderpatients was performed after short term in vitro culture. Patient QUIwas excluded since no normal renal cells have been obtained. All the sixother patients expressed high level of MHC class I molecule on bothnormal and tumoral cells and were positive for the adhesion moleculeCD54. As expected, G250 was specifically expressed on tumoral cells butat various levels. The marker AS02 indicated the level of fibroblasticcontamination in the culture. That ranged from 4.7% to 58.3% for thenormal cells and from 6.7% to 53.2% for the tumoral counterparts for thepatients BEL, MOR, POU,VAG and ZEN. It is important to note that exceptfor ZEN, the fibroblast (which is a cell type resistant to γδ T celllysis) contamination level is higher in the tumoral culture that in thecorresponding normal cell culture. Patient FOUR showed a largecontamination, since in the tumoral culture only the fibroblasts weregrowing (98.5% of AS02 positive cells). Patient FOUR was then alsoexcluded from the main part of the study. MICA expression level, readout by BAM195 staining, is rather low on all the cells tested, exceptfor BEL normal renal cells.

In vitro Cytotoxicity of Expanded Vγ9Vδ2 T Cells from mRCC Patients

Lytic activities of amplified γδT cells were measured against classicalcontrol targets (Raji and Daudi), primary autologous normal and tumorcell lines of the selected patients in a 4 h standard ⁵¹Cr release assayfor the five patients BEL, MOR, POU,VAG and ZEN. Individual data and themean of these five patients results are shown in FIGS. 9A and 9B.

-   -   Vγ9Vδ2 T cells expanded by BrHPP exhibited 36.7±5.4% (range 29.1        to 43.1%) of specific lysis against primary tumor cell lines for        the 30:1 ratio, whereas the primary normal cell lines were lysed        at 12.5±5.0% (range 7.0 to 18.9%) at the same ratio. These        results are the mean of the five patients tested.    -   The lysis level of tumoral cells by autologous effector cells is        significantly higher compared to the normal control. In a        bidirectional paired student's t test, the p value is: p=0.0002        at 30:1 E/T ratio, p=0.01 at 15:1 E/T ratio and p=0.04 at 7.5:1        E/T ratio.

In all cultures analyzed, γδ effectors T cells presented a strong lyticactivity against control sensitive target cell line Daudi (68.3±14.2%)and very low activity against control negative target cell line Raji(12.9±5.0%/0).

For patient ZEN, lysis by autologous LAK cells was investigated. Asexpected LAK cells showed a lytic activity against Daudi and Raji celllines but also the normal and tumoral cells with compared efficiency.These results have to be confirmed with other patients but theyunderline the lack of specificity of LAK cells mediated lysis.

8.3 Discussion

In this study, we have shown that peripheral Vγ9Vδ2 T cells from RCCpatients can be expanded in vitro by BrHPP stimulation in 58% of thecases (7 patients out of 12). These cells displayed a selective lysisagainst autologous renal tumor cells and not against renal normal cellsas read out by cytotoxic assay.

However, this lytic activity of expanded γδ T cells toward primary tumorcells may have been minimized due to a variable level of contaminationby fibroblasts in these cultures: the percentages of fibroblasts rangedfrom 4.7% to 58.3% for normal cells and ranged from 6.7% to 53.2% fortumor cells. This contamination of primary cell culture modified the E/Tratio of the cytotoxic assay.

In order to realize this test in better conditions, we proposed to sortthe fibroblasts with the specific antibody (clone AS02, Dianova) andthen to culture the purified renal tumor cells.

We have obtained confirmation that expanded γδ T cells did not lysefibroblasts. The culture of tumor cells from one patient of this study(patient FOUR) presented 98.5% of contamination by fibroblasts. Thecytotoxic assay with the expanded autologous Vγ9Vδ2 T cells showed thatthese cells were lysed at 4.0% at the 30:1 ratio whereas Daudi werelysed at 75.4% at the same ratio.

To conclude, this study showed that for all the evaluable patients theprimary tumor cells but not normal cells could be specifically lysed byautologous in vitro BrHPP stimulated γδ T cells.

Example 9 In vitro and in vivo Dosage Response Comparison of BrHPP andHDMAPP Compounds

9.1 Materials and Methods

9.1.1 In vivo

Animals

Eight purpose bred healthy male cynomolgus monkeys (M. fascicularis). Atthe beginning of the study, body weights ranged from 3.5 to 4 kg andages from 37 to 41 months.

Husbandry conditions conformed to the European requirements, comprisingmonitored temperature, humidity, air change and lighting cycle. Animalswere housed individually in stainless steel cages. Food was provided adlibitum and composed of expanded complete primate diet (U. A. R.,Villemoisson, Epinay/Orge, France) supplemented daily with fresh fruits.Animals were anaesthetised with intramuscular injection of 6 mg/kgZoletil™ 100 (Tiletamine-Zolazepam, Virbac, Carros, France) before anyperfusion.

HDMAPP/IL2

HDMAPP: initially at 21.5 mM.

HDMAPP was produced according to the methods described herein. It wassterilized by filtration on 0.22 μM microfilters. HDMAPP solutions inwater are stored frozen

IL2: Proleukin® from Chiron (Emeryville, USA), at 18M UI per vial,stored at −20° C., reconstituted with 1 ml sterile water for injection.This starting solution at 18M IU/ml is diluted qsp 10 ml water, anddoses of 300 μl (0.6M IU) are injected daily. Each diluted solutionbatch is stored at 4° C. for up to 3 days.

Injections/Blood Samplings

HDMAPP was administered i.v. to male cynomolgus monkeys by 30-minuteperfusions in a total volume of 50 ml with Ringer Lactate as vehicle.

Injected HDMAPP doses: 2.5 mg/kg, 0.5 mg/kg, 0.1 mg/kg and 0.02 mg/kg.

The animals were co-treated subcutaneously with 0.6M IU IL2 in sterilewater per day for 5 days.

Blood was drawn pre-dose and at day 4, 5, 7, 11 and 14 after HDMAPPperfusion for flow cytometry analysis of blood cellular populations ofinterest.

Blood samples (1 to 4 ml) were withdrawn from femoral vessels/arteryinto heparin-lithium containing tubes. Tubes were shipped overnight atroom temperature (RT) before flow cytometry analyses.

Flow Cytometry

Blood samples (1 to 4 ml) were withdrawn from femoral vessels/arteryinto heparin-lithium containing tubes. Tubes were shipped overnight atroom temperature (RT) before flow cytometry analyses.

Peripheral γδ lymphocytes were analysed by flow cytometry on totalmonkey blood, after triple staining with anti-Vgamma9FITC, anti-CD3PEand anti-CD69PC5 antibodies (Vgamma9-FITC: 7B6 clone, produced, purifiedand FITC-coupled at Innate Pharma; CD3-PE : SP34 clone, BD BiosciencesPharmingen, Le Pont de Claix, France; CD69PC5: FN50 clone,Immunotech-Beckman-Coulter, Marseilles, France).

Briefly, 50 μl monkey blood was incubated 15 min at RT with 10 μlanti-gamma9-FITC, 5 μl anti-CD3-PE and 5 μl anti-CD69PC5 antibodies.Antibodies were washed with 3 ml 1× PBS, centrifuged for 4 min at 1300rpm at RT and supernatant was discarded. Red cells were lysed with theOptiLyse C reagent (Immunotech-Beckman-Coulter, Marseilles, France)according to the manufacturer's instructions. At the final step, stainedwhite blood cells were recovered by centrifugation and resuspended in300 μl 1× PBS+0.2% PFA. Immediately before analysis, 50 μl calibratedFlow Count™ Fluorospheres (Immunotech-Beckman-Coulter, Marseilles,France) were added to the cells for absolute number counting of thepopulations of interest.

Flow cytometry was performed on a Epics XL-MCL apparatus(Beckman-Coulter, Roissy, France) with the Expo32 software.

9.1.2 In vitro

In vitro proliferation assays and in vitro TNFα release assay areperformed essentially as described in Example 3.

9.2 Results

9.2.1 In vivo

HDMAPP injections on the 8 test animals were carried out according tothe following schedule in Table 2: TABLE 2 Day 20 of Day 31 of Day 18 ofDay 30 of Animals\Dates month 1 month 2 month 4 month 5 AD235 2.5 mg/kg2.5 mg/kg 2.5 mg/kg 2.5 mg/kg* AD384 0.5 mg/kg 0.5 mg/kg AD101 0.1 mg/kg0.1 mg/kg AC903 2.5 mg/kg 0.5 mg/kg AD270 0.5 mg/kg 0.5 mg/kg AD299 0.1mg/kg 0.02 mg/kg AD602 0.02 mg/kg AD219 0.02 mg/kg*The result of this injection was not used for the calculation of thedose-range effect (see below).

The effect of successive i.v. injections of various HDMAPP doses tocynomolgus monkeys was monitored. Individual curves of percentages ofperipheral γδ T cells and absolute blood count of the same cells wasdetermined for each animal.

In vivo amplification was assessed after 4 successive injections, 5weeks apart, of 2.6 mg/kg HDMAPP. The 4 successive HDMAPP treatments ledrespectively to approx. 60, 55, 60 and 40% γδ cells among CD3⁺ at day5).

In vivo amplification was assessed after 4 successive injections of 20or 80 mg/kg Phosphostim®, 4 or 8 weeks apart. The 4 successive BrHPPtreatments led respectively to repeated increases of γδ cells asdescribed in previous examples herein.

The dose-range effect of HDMAPP and BrHPP in vivo as determined bydetermining numbers of γδ T cells by flow cytometry are shown in FIGS.10 to 13. HDMAPP was tested in four doses as described in Table 2, andas shown, these data were obtained from various numbers of injections(2.5 mg/kg: 3 injections; 0.5 mg/kg: 5 injections; 0.1 mg/kg: 3injections; 0.02 mg/kg: 3 injections). BrHPP was tested in seven doses:0, 0.12, 2.4, 12, 48, 72 and 96 mg/kg. The figures present γδ T cellscounted at days 5 and 7 for HDMAPP and day 7 for BrHPP. As discussedabove, the peak of γδ T cell expansion is found to be at day 5 afterinjection. For each dose range, results are expressed in (a) fold γδ Tcell increase in absolute cell count (/mm3 blood), (b) absolute cellcount (/mm³ blood), (c) percentage γδ T cells of total circulatinglymphocytes, and (d) fold increase in percentage of total circulatinglymphocytes.

FIGS. 10A and 10B show the absolute cell count (/mm³ blood) for HDMAPPand BrHPP respectively.

FIGS. 11A and 11B show the percentage γδ T cells of total circulatinglymphocytes for HDMAPP and BrHPP respectively.

FIGS. 12A and 12B show the fold γδ T cell increase in absolute cellcount (/mm³ blood) for HDMAPP and BrHPP respectively.

FIGS. 13A and 13B show the fold increase in percentage of totalcirculating lymphocytes for HDMAPP and BrHPP respectively.

9.2.1 Comparison of BrHPP and HDMAPP in vitro and in vivo Bioactivity

BrBPP and HDMAPP bioactivity were compared using in vitro and in vivoassays. The in vitro biological activity of γδ cell amplification fromhuman PBMCs (in the presence of rhIL2) was assessed using a TNFα releaseassay. In vivo activity was determined as described above and shown inFIGS. 10 to 13. For purposes of comparison, the aminobisphosphonatecompound Zoledronate®, the most potent aminobisphosphonate evaluated sofar by the inventors, was included in the in vivo comparison.

The in vitro comparison is shown in FIG. 14. FIG. 14 shows the in vitroEC50 for the compounds: for BrHPP the EC50 is about 30 nM while forHDMAPP the EC50 is about 0.6 nM, approximately a 50-fold difference inpotency.

The in vivo comparison is shown in FIG. 15. FIG. 15 shows the in vivoEC50 for the compounds: for BrHPP the EC50 is about 1 nM while forHDMAPP the EC50 is about 5 pM, approximately a 2-log difference inpotency. By contrast, the less potent Zoledronate® showed an EC50 valueof about 1 μM.

Example 10 In vivo Efficacy of Human γδ T Cells Nod-Scid/Tumor Model

The aim of this project based on the use of the Nod-SCID/tumor micemodel was to study the proliferation of human γδ T cells after an invivo single stimulation of human MNC with BrHPP and to study the in vivoanti-tumoral efficacy of the developed γδ T cells.

Materials and Methods

Preliminary studies to set up the Nod-SCID/tumor and to stimulate humanMNC with BrHPP in the Nod-SCID were carried out as follows:

The following renal tumor cells lines purchased from the ATCC (786-O,Kacil, G401 and G402) were used for the establishment of the model.786-0 and Kaci-1 cells lines were renal clear cell carcinoma (RCC), G401rhabdoid and G402 leimyeloblastoma 8-12 weeks Nod-SCID mice were used asrecipient of the cells lines.

Each mouse received, at the left flank's upper part, 2-5×10⁶ cells bysubcutaneous injection at day D0. The appearance and the growing of thetumors evolution were checked weekly.

The tumors appearance and development depend on the cell line and on thenumber of engrafted cells. For example, after the engraftment of 2×10⁶cells, 3 to 4 weeks were needed for the 786-O and Kaci-1, while 2 weekswere needed for the G401 and 402.

At day 0, mice were injected IP 50*10⁶ PBMC (collected from healthydonor, établissement francais du sang), then stimulated (IP) 40 mg/kgBrHPP mixed with 1000 IU of IL2. At D5 and each every three days, micewere treated with 500 UI of IL2. Human γδ T cells were developed in theperitoneal cavity of Nod-SCID/human (hu) BrHPP treated mice.

Anti-Tumoral Efficacy

Taking advantage of the tumoral development in the Nod-SCID mice and thein vivo stimulation of the human γδ T cells, the anti-tumoral effect ofthe γδ T cells was studied in vivo. The tumoral model was established byengrafting 2×10⁶ of 786-0 cells lines, three weeks later, when thevolume of the solid tumor reached more than >30 mm3 (calculated with theformula A²× B/2 where A and B represent respectively the length andbreadth of the tumor). Mice, randomized, received IP PBMC and treatedfor the stimulation with BrHPP. Several groups were constituted:

A—Negative control group: Mice in the group received only tumoral cellline

B—PBMC group: Mice were injected IP with 50×10⁶ PBMC

C—Study group: Mice after receiving the 50×10⁶ PBMC were treated withBrHPP and IL2.

For each group, parameters were observed as follows:

Weekly check of the tumor size and volume

Weekly phenotype of human T cells in the peritoneal cavity and bloodcollected cells for the percentage of human γδ T cells versus ab human Tcells.

Serological TNF and INF check

At sacrifice: 1-phenotype study of the homing of human leukocytes cellsand their phenotype (IP, liver, lung, spleen, bone morrow, blood, thymusand tumor).

-   -   2-Immunohistochemistry (IHC) study in the tumor of the recipient        mice.        Results    -   Tumor growth: Human IL2 treatment of the 786-O tumor did not        induce any activity; no difference was observed between the        tumor size in this group and the non treated mice group.        -   Tumor growth in the first few days after BrHPP treatment is            shown in FIG. 15. While tumor size increased in the first            few days after treatment, tumor size decreased therafter            quickly in the PBMC/BrHPP and IL2 treated group. FIG. 19            shows that from day 7 onwards the tumor size shrank. In the            PBMC group, after short arrest, the size grows and no            significant difference was observed between the tumors sizes            in this group and those of the negative control.    -   Phenotyping and homing of human cells: In the peritoneal cavity:        the weekly check of the 1P phenotype of treated mice showed a        human to γδ T cell presence only in the BrHPP treated mice. The        relative numbers of γδ T cells in the blood is shown in FIG. 16        and in the peritoneal cavity in FIG. 17. Human γδ T cells        represent a higher percentage of the human CD3 T cells. In the        mice recipient organs: phenotyping is carried out at sacrifice        (4 weeks after the PBMC and BrBPP treated or not treated        groups); the major human cells present in those organs are human        CD3+ T cells with 99% expression of the Ab TcR. However in the        tumor, human γδ cells were present only in the BrHPP treated        group.    -   IHC: 4 μm slides of the tumor collected at the sacrifice        confirmed the γδ T cells presence only in the tumors from PBMC        BrHPP treated mice. The frequency of the human γδ T cells inside        the tumor looks to be correlated with the delay of the BrHPP        treatment.        Conclusion

Data obtained here demonstrated the γδ efficacy in vivo. γδ T cellsexercise their activity in the 2 weeks after the BrHPP stimulation. Thiseffect is prolonged and stable, in the group of mice where the tumor wascleared, no reappearance of new tumor was observed 12 weeks after thetreatment. In 4 of 10 mice, the tumor was not totally cleared; but evenin this group, tumor growth was stopped and the tumor size stayed stableafter 12 weeks. The TIL in the BrHPP-untreated mice consists of ab Tcells; however we did not observe any tumoral activity in this group.

Example 11 Administration of BrHPP for Treatment of Advanced/MetastaticSolid tumors in Humans

Phosphostim is based on a new chemical entity, the drug substanceBromohydrin Pyrophosphate (BrHPP), which is a specific agonist of immunecompetent cells namely the Vγ9Vδ2 T cell subpopulation bearinganti-tumor activity. Phosphostim (BrHPP, 200 mg) is the intravenousformulation of BrHPP for cancer immunotherapy, which will be used incombination with low dose of IL-2 (s.c. 1 M IU/m²/day) in a firstclinical trial in patients with advanced/metastatic solid tumors.

Clinical Trial

Phosphostim has never been previously administered to humans. The mainobjective of the planned phase I clinical trial with Phosphostim is toevaluate the safety and tolerance of a Phosphostim alone and incombination with a fixed dose of IL-2. This trial is a single arm,open-label, national, multi-center, dose-escalation trial in sequentialcohorts of patients with advanced/metastatic solid tumors. A traditionaldose escalation design (Fibronacci) will be used, with cohorts of 36patients at each dose level. All patient cohorts will receive repeatedcycles of treatment every 3 weeks. The first cycle will consist of oneadministration by infusion of Phosphostim alone and from the secondcycle onwards, 1 million IU/m²day of IL-2 will be added (for a totalduration of 7 days). Phosphostim starting dose will be 200 mg/m² (5mg/kg) corresponding to 118 mg-equivalent of BrHPP anionic form. Thepharmaco-kinetics and pharmaco-dynamics properties of Phosphostim aloneand in combination with IL-2 will also be evaluated in this study.

Phosphostim (BrHPP, 200 mg) is a freeze-dried apyrogenic sterile whitepowder to be reconstituted in solution for infusion.

Each vial of Phosphostim (BrHPP, 200 mg) contains 200 mg of BrHPPanionic form and 50 mg the excipient alpha-lactose monohydrate (USP).

Phosphostim (BrHPP, 200 mg) has a shelf life of 6 months at −20° C.Additional stability studies are currently on-going. Until these studiesare completed, Phosphostim should be stored at −20° C. ±5° C., protectedfrom light.

Phosphostim is for immediate and single use following first opening andreconstitution. Phosphostim is reconstituted immediately prior to usewith 2 ml of water for injections to make a 100 mg/ml solution. Theneeded quantities of reconstituted product are diluted in a total volume100 ml of ringer lactate buffer infusion vehicle. The diluted solutionis clear and colorless.

Phosphostim is administered intravenously over 1 hour.

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1-79. (canceled)
 80. A method of treating a disease comprising theadministration of a composition γδ cell activator comprising apharmaceutically acceptable carrier in an amount sufficient to induce anat least 5-fold increase in the γδ T cell population in a subject,wherein said disease is selected from the group consisting of cancer,solid tumors, infectious diseases, autoimmune diseases and allergicdisease.
 81. The method according to claim 80, wherein said γδ T cellactivator is provided in an amount sufficient to induce an at least10-fold increase in the γδ T cell population in a subject.
 82. Themethod according to claim 80, wherein at least two treatments areadministered to said subject.
 83. The method according to claim 80,wherein at least four treatments are administered to said subject. 84.The method according to claim 80, wherein the γδ T cell activator isadministered in more than one treatment with an interval of about two toabout eight weeks between treatments.
 85. The method according to claim80, wherein the γδ T cell activator is administered in more than onetreatment with an interval of about three to about four weeks betweentreatments.
 86. The method according to claim 80, wherein said γδ T cellactivator is provided in an amount sufficient to expand the γδ T cellpopulation in a subject to reach between 30-90% of total circulatinglymphocytes in a subject.
 87. The method according to claim 80, whereinthe biological activity of γδ T cells are increased in said subject. 88.The method according to claim 80, wherein the solid tumor is renalcancer.
 89. The method according to claim 80, wherein said solid tumoris selected from the group consisting of a melanoma, ovarian cancer,colon cancer, lung cancer, pancreatic cancer, neuroblastoma, head orneck cancer, bladder cancer, breast cancer, brain cancer and gastriccancer.
 90. The method according to claim 80, wherein the γδ T cellactivator is a composition comprising a compound capable of inducing theproliferation of a γδ T cell in a pure population of γδ T cell cloneswhen said compound is present in culture at a concentration of less than1 mM.
 91. The method according to claim 80, wherein the γδ T cellactivator is a compound of formula (I):

wherein Cat+ represents at least one organic or mineral cation that canbe the same or different; m is an integer from 1 to 3; B is O, NH, orany group capable of being hydrolyzed; Y═O⁻Cat+; a C₁-C₃ alkyl group;-A-R; or a radical selected from the group consisting of a nucleoside,an oligonucleotide, a nucleic acid, an amino acid, a peptide, a protein,a monosaccharide, an oligosaccharide, a polysaccharide, a fatty acid, asimple lipid, a complex lipid, a folic acid, a tetrahydrofolic acid, aphosphoric acid, an inositol, a vitamin, a co-enzyme, a flavonoid, analdehyde, an epoxyde and a halohydrin; A is O, NH, CHF, CF₂ or CH₂; and,R is a linear, branched, or cyclic, aromatic, non-aromatic, saturated orunsaturated C₁-C₅₀ hydrocarbon group, optionally interrupted by at leastone heteroatom, wherein said hydrocarbon group comprises an alkyl, analkylenyl, an alkynyl or an alkylene, which can be substituted by one orseveral substituents selected from the group consisting of: an alkyl, analkylenyl, an alkynyl, an epoxyalkyl, an aryl, an heterocycle, analkoxy, an acyl, an alcohol, a carboxylic group (—COOH), an ester, anamine, an amino group (—NH₂), an amide (—CONH₂), an imine, a nitrile, anhydroxyl (—OH), a aldehyde group (—CHO), a halogen, a halogenoalkyl, athiol (—SH), a thioalkyl, a sulfone, a sulfoxide, and a combinationthereof.
 92. The method according to claim 91, wherein the γδ T cellactivator is a compound of formula (II):

in which X is an halogen, B is O or NH, m is an integer from 1 to 3, R1is a methyl or ethyl group, Cat+ represents at least one organic ormineral cation, n is an integer from 2 to 20, A is O, NH, CHF, CF₂ orCH₂, and Y is O⁻Cat+, a nucleoside, or a radical -A-R, wherein R isselected from the group consisting of:

wherein n is an integer from 2 to 20, R₁ is a (C₁-C₃)alkyl group, and R₂is an halogenated (C₁-C₃)alkyl, a (C₁-C₃)alkoxy-(C₁-C₃)alkyl, anhalogenated (C₂-C₃)acyl or a (C₁-C₃)alkoxy-(C₂-C₃)acyl;

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group;and

wherein R₃, R₄, and R₅ are identical or different and are a hydrogen or(C₁-C₃)alkyl group, W is —CH— or —N— and R₆ is an (C₂-C₃)acyl, analdehyde, an (C₁-C₃)alcohol, or an (C₂-C₃)ester.
 93. The methodaccording to claim 92, wherein the compound of formula (II) is (R,S)-3-(bromomethyl)-3-butanol-1-yl-diphosphate.
 94. The method accordingto claim 92, wherein the γδ T cell activator is administered in a doseto humans between 10 mg/kg to 100 mg/kg.
 95. The method according toclaim 92, wherein said γδ T activator is administered by intravenousinfusion in a dose to humans that is calculated according to the formula(I): single dose (mg/kg)=(10 to 100)*N (I), where N is the number ofweeks between treatments such that N is between about 3 and about
 4. 96.The method according to claim 91, wherein the γδ T cell activator is acompound of formula (XII):

in which R₃, R₄, and R₅ are identical or different and are a hydrogen or(C₁-C₃)alkyl group, W is —CH— or —N—, R₆ is an (C₂-C₃)acyl, an aldehyde,an (C₁-C₃)alcohol, or an (C₂-C₃)ester, Cat+ represents at least oneorganic or mineral cation that can be the same or different, B is O orNH, m is an integer from 1 to 3, A is O, NH, CHF, CF₂ or CH₂, and Y isO⁻Cat+, a nucleoside, or a radical -A-R, wherein R is selected from thegroup consisting of:

wherein n is an integer from 2 to 20, R₁ is a (C₁-C₃)alkyl group, and R₂is an halogenated (C₁-C₃)alkyl, a (C₁-C₃)alkoxy-(C₁-C₃)alkyl, anhalogenated (C₂-C₃)acyl or a (C₁-C₃)alkoxy-(C₂-C₃)acyl;

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group;and

wherein R₃, R₄, and R₅ are identical or different and are a hydrogen or(C₁-C₃)alkyl group, W is CH or N, and R₆ is an (C₂-C₃)acyl, an aldehyde,an (C₁-C₃)alcohol, or an (C₂-C₃)ester.
 97. The method according to claim96, wherein the compound of formula (XII) is(E)-4-hydroxy-3-methyl-2-butenyl pyrophosphate.
 98. The method accordingto claim 96, wherein the compound of formula (XII) is(E)-5-hydroxy-4-methylpent-3-enyl pyrophosphonate.
 99. The methodaccording to claim 96 where said γδT activator is administered byintravenous infusion in a dose to humans that is calculated according tothe formula (I) single dose (mg/kg)=(0.01 to 10)*N (I) where N is thenumber of weeks between treatments such that N is between about 3 andabout
 4. 100. The method according to claim 80, further comprisingseparately administering to a subject in need thereof an effectiveamount of a γδT activator and an interleukin-2 polypeptide.
 101. Themethod according to claim 100, wherein the interleukin-2 polypeptide isadministered over a period of time comprised between 1 and 10 days.